Methods of treating or preventing rheumatic disease

ABSTRACT

The present disclosure provides a method for treating or preventing a rheumatic disease, comprising administering a population of cells enriched for STRO-1 +  cells and/or progeny thereof and/or soluble factors derived therefrom.

RELATED APPLICATION DATA

This application is a continuation of U.S. Ser. No. 14/992,487, filedJan. 11, 2016, now allowed, which is a continuation of U.S. Ser. No.14/130,647, filed May 20, 2014, now U.S. Pat. No. 9,265,796, issued Feb.23, 2016, which is a § 371 national stage of PCT InternationalApplication No. PCT/AU2012/000799, filed Jul. 4, 2012, which claimspriority of Australian Patent Application No. 2011902655, filed Jul. 4,2011, the entire contents of each of which are hereby incorporated byreference into this application.

SEQUENCE LISTING REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named “180417 85874-AASubstitute Sequence Listing CAE.txt”, which is 7.41 kilobytes in size,and which was created Apr. 17, 2018 in the IBM-PC machine format, havingan operating system compatibility with MS-Windows, which is contained inthe text file filed Apr. 17, 2018 as part of this application.

FIELD

The present invention relates to methods for treating or preventingrheumatic diseases.

BACKGROUND

Rheumatic diseases are a class of common diseases generally called“arthritis”, which are often associated with or caused by an autoimmuneresponse. This class of diseases includes rheumatoid arthritis,spondyloarthropathies (e.g., ankylosing spondylitis), Sjorgren Syndrome(Sicca Syndrome), Reiter's disease, psoriatric arthritis, entericarthritis (joint problems associated with inflammatory bowel disease),sacroiliitis or spondylitis), and osteoarthritis.

According to the Center for Disease Control (CDC), approximately 50million adults have been diagnosed with some form of arthritis in USAalone. This number is predicted to increase to 67 million adults by2030. The CDC estimates that the cost of arthritis in USA wasapproximately US$80.8 billion for treatments for arthritis andapproximately US$47 billion in indirect costs (e.g., lost earnings). Thetotal cost $127.8 billion was 1.2% of the US gross domestic product in2003.

Rheumatoid Arthritis and Osteoarthritis

Rheumatoid arthritis (RA) is a painful chronic systemic diseasecharacterized by extensive synovial inflammation accompanied bydestruction of joint cartilage and bone. The clinical course of RA isvariable and often shows a remitting pattern but if progressiveinevitably leads to joint deformity and impaired function. Three formsof RA can be distinguished: mild, self-limiting disease; mildlyprogressive disease; and aggressive disease which is difficult tocontrol with medication, and is characterized by functional decline andradiologic deterioration of the joints, e.g., joint space narrowing andcartilage erosions, particularly beneath the proliferating inflamedsynovium referred to as pannus. In accordance with the systemic natureof the disease, there are extra-articular manifestations which includevasculitis, alveolitis, and ocular disease. Prevalence of the disease asreported in the literature is approximately 1% of the U.S. population,with women accounting for two-thirds of all cases.

The onset of RA is often insidious with fatigue, anorexia, generalizedweakness, and musculoskeletal pain. Specific symptoms appear later.Several joints, usually in a symmetrical fashion, are affected. Mostoften these are joints of the hands, wrists, knees, and feet. Joints arepainful and swollen, and motion is limited. Morning stiffness of morethan one hour is a very typical finding. With persistent inflammation, avariety of deformities develop which include most typically radialdeviation of the wrist and hyperextension or flexion of the proximalinterphalangeal joints; other deformities occur as well. Atrophy ofskeletal muscle sets in. In approximately 20 to 30% of all patients,there is development of rheumatoid nodules on periarticular structuresor sites of trauma, but they are usually of limited clinicalsignificance. The nodules may be found in other structures such as thepleura or the meninges. Rheumatoid vasculitis can affect nearly allorgan systems (lung, gastro-intestinal-tract, liver, spleen, pancreas,lymph nodes, testis, and the eye).

There is no curative treatment for RA. All drug regimens primarilyattempt to relieve the symptoms and the inflammation. Aspirin and othernonsteroidal anti-inflammatory drugs (NSAIDs) with a rapid onset ofaction are the first line of treatment. Oral and injectableglucocorticoids are added to the drug regimen if necessary. The thirdline of treatment includes disease modifying antirheumatic drugs(DMARDs); they have a slow onset of action, in some cases severalmonths. DMARDs include azathioprine, sulphasalazine, gold,D-penicillamine, hydroxychloroquine, methotrexate, and cyclosporine. Themore recent addition of biological drugs, such as Enbrel®, Remicade® andHumira® has provided an alternative mode of therapy, however regular useof these products can suppress the immuno defence system and has beenassociated with increased incidence of opportunistic infections anddiseases such as tuberculosis.

Osteoarthritis (OA) is the most common form of arthritis in Westernpopulations. Knee OA, characterized clinically by pain and functionaldisability, is the leading cause of chronic disability among the elderlyin the US.

Pathologically, the most striking changes in OA are focal loss ofarticular cartilage and marginal and central new bone formation.However, OA is not simply a disease of articular cartilage and thesubchondral bone. Rather, it is a disease of the synovial joint, withalterations also found in the synovium, capsule, ligaments,periarticular muscle, and sensory nerves.

Although OA was once considered a non-inflammatory arthropathy, patientsoften present with signs and symptoms consistent with local inflammationand synovitis, and recent evidence from preclinical and clinical studiessupports the role of inflammation and inflammatory mediators in itspathophysiology.

Current treatment of osteoarthritis includes non-medicinal therapy,medicinal therapy, and surgical treatments. Non-medicinal treatmentsinclude exercise, and weightloss, programs, thermal treatment, andassistive devices or bracing. For knee OA, range-of-motion andstrengthening exercises are geared toward reduction of impairment,improvement of function, and joint protection. Medications includeanalgesics (e.g., acetaminophen), non-steroidal anti-inflammatory drugs(NSAIDS) that are either non-selective cyclooxygenase (COX) inhibitorsor selective inhibitors of the COX-2 enzyme, injected intra-articularcorticosteroids or viscosupplementation, and proven or putativedisease-modifying osteoarthritis drugs (DMOADs). Surgical proceduresinclude joint debridement and lavage, and lastly total kneearthroplasty.

The most commonly used medicinal treatments for knee OA typicallyprovide less than 50% relief of pain. For example, use of acetaminophen,selective NSAIDs or non-selective NSAIDs typically results in meanimprovements in knee OA pain of no more than 30 points from a baselineof about 70 points using 100 point (100-mm) visual analog scales. Thus,there is substantial room for improvement in the pain management of kneeOA. Further, no therapy has been demonstrated to retard the progressionof structural degradation.

Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic, progressive, inflammatorydisease with considerable impact on patient functioning, well-being, anddisability. The prevalence of AS has traditionally been estimated in therange of 0.1-1.9%, with more males affected than females As a chronicdisease of the axial skeleton and large peripheral joints, AS causesinflammatory back pain and stiffness and it is associated with otherinflammatory diseases of the skin, eyes and intestines. In severe cases,AS may result in complete spinal fusion, causing extreme physicallimitation. Thus, there remains a need for a safe and effectivetreatment for AS.

As the disease progresses, patients with AS experience pain, jointstiffness, and the eventual loss of spinal mobility. These clinicalsymptoms and subsequent disease progression result in functionallimitations and impairment in health-related quality of life (HRQOL).

No cure exists for AS. Generally, treatment includes trying to relievepain and stiffness using medications such as NSAIDs, corticosteroids,and DMARDs.

It will be apparent to the skilled artisan that inflammatory jointdisease is a class of debilitating diseases having a major impact onsociety. There is also a need for therapeutics for these diseases.

SUMMARY

In work leading up to the present invention, the inventors sought todetermine the effect of mesenchymal progenitor cells (MPCs) on rheumaticdiseases. The inventors studied a sheep model of rheumatoid arthritis asa model for rheumatic diseases generally, since many of these diseasesshare common features, e.g., autoimmunity and the presence of immunecells and inflammatory cytokines in the joint. The inventors havedetermined that MPCs were effective in reducing histopathologicalindices of arthritis, such as, synovial hyperplasia, stromal tissueactivation and inflammatory cell infiltration. Furthermore, theinventors have shown that administration of MPCs reduces levels ofpro-inflammatory cytokines, such as, IL-6, TNFα, IL-17 in addition toCD14⁺ cells, e.g., in synovial tissue. The inventors consider that theability to reduce levels of these cells and cytokines make MPCs (and/ortheir progeny and/or soluble factors secreted therefrom) suitable fortreating rheumatic diseases, such as, rheumatoid arthritis and/orosteoarthritis.

The inventors have also determined that MPCs provide a prolongedtherapeutic benefit, with the effects of a single injection of cellslasting for at least about 30 days. The inventors have additionallyfound that MPCs or progeny thereof migrated to the site of pathology ofthe rheumatic disease. Such migration provides benefits since it permitssystemic administration of the cells, which is both easier and can beless difficult and/or less invasive, e.g., compared to administeringcells into synovial fluid of a subject.

Ovine MPCs isolated by expression of the marker STRO-3 are functionallyequivalent to human MPCs which co-express the markers STRO-3 and STRO-1(as shown herein in Example 2).

The present disclosure therefore provides a method for treating orpreventing an rheumatic disease in a subject, the method comprisingadministering to the subject a population of cells enriched for STRO-1⁺cells and/or progeny thereof and/or soluble factors derived therefrom.

In one example, the rheumatic disease is an autoimmune rheumatic disease

In one example, the rheumatic disease is selected from the groupconsisting of rheumatoid arthritis, Still's disease (syn. juvenileidiopathic arthritis or juvenile rheumatoid arthritis), ankylosingspondylitis, Reiter's disease, psoriatric arthritis, enteric arthritis,sacroiliitis, spondylitis and osteoarthritis.

In one example, the rheumatic disease is osteoarthritis.

In an exemplary form, the rheumatic disease is rheumatoid arthritis.

In one example, the method comprises administering a population of cellsenriched for STRO-1^(bright) cells and/or progeny thereof and/or solublefactors derived therefrom.

In one example, the population enriched for STRO-1⁺ cells and/or progenythereof and/or soluble factors derived therefrom are administeredsystemically. For example, the cells are administered intravenously. Inthis regard, the inventors have shown that STRO-1⁺ cells and/or progenythereof migrate to the site of an inflamed joint in a subject. Thus, thedisclosure contemplates administration of the STRO-1⁺ cells and/orprogeny thereof at a site remote from an inflamed joint in a subject.

In one example, the STRO-1⁺ cells and/or progeny thereof and/or solublefactors derived therefrom are administered in an amount sufficient toreduce IL-6, TNFα, IL-17 in addition to CD14⁺ cells in a subject, e.g.,within a joint of a subject, such as within synovial tissue.

Exemplary dosages of the cells include between 0.1×10⁶ to 5×10⁶ STRO-1⁺cells and/or progeny thereof. For example, the method comprisesadministering between 0.3×10⁶ to 2×10⁶ STRO-1⁺ cells and/or progenythereof per kilogram.

In one example, the cells are administered at a dose of between about0.3×10⁶ cells/kg to about 4×10⁶ cells/kg, such as between about 0.3×10⁶cells/kg to about 2×10⁶ cells/kg.

One form of the method involves administering a low dose of STRO-1⁺cells and/or progeny thereof. Such a low dose is, for example, between0.1×10⁵ to about 0.5×10⁶ STRO-1⁺ cells/kg, such as about 0.3×10⁶ STRO-1⁺cells/kg.

In another example, a high dose of cells is administered to the subject.Exemplary dosages include at least about 1.5×10⁶ cells/kg. For example,a high dose comprises between about 1.5×10⁶ to about 4×10⁶ cells/kg. Forexample, a high dose comprises about 1.5×10⁶ or about 2×10⁶ cells/kg.The inventors have shown that such doses provide benefits, e.g., byreducing levels of IL-6, TNFα, IL-17 in addition to CD14⁺ cells.

In one example, the cells are administered at a dose of about 100million to 300 million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 100million to 200 million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 100million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 150million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 200million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 300million cells irrespective of the weight of the patient.

In one example, the population enriched for STRO-1⁺ cells and/or progenythereof and/or soluble factors derived therefrom are administered onceweekly or less often, such as, once every four weeks or less often.

The present disclosure also contemplates numerous administrations of thecells and/or soluble factors. For example, such a method can involveadministering the cells and monitoring the subject to determine when oneor more symptoms of an inflammatory joint disease occurs or recurs andadministering a further dose of the cells and/or soluble factors.Suitable methods for assessing symptoms of an rheumatic disease will beapparent to the skilled artisan and/or described herein.

In another example, cells and/or soluble factors are administered on afixed schedule, e.g., once each week or fortnight or three weeks or fourweeks or five weeks or six weeks or longer.

In one example, the population enriched for STRO-1⁺ cells and/or progenycells are autogeneic or allogeneic and/or the soluble factors can bederived from autogeneic or allogeneic cells.

In one example, the population enriched for STRO-1⁺ cells and/or progenycells have been culture expanded prior to administration and/or prior toobtaining the soluble factors.

In one example, the population enriched for STRO-1⁺ cells areSTRO-1^(bright), and/or express tissue non-specific alkaline phosphatase(TNAP) and/or the progeny cells and/or soluble factors are derived fromSTRO-1⁺ cells that are STRO-1^(bright) and/or express TNAP.

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered in the form of acomposition comprising said STRO-1⁺ cells and/or progeny cells thereofand/or soluble factors derived therefrom and a carrier and/or excipient.

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered together with anothercompound for treating or preventing an rheumatic disease. In oneexample, the other compound is a disease-modifying anti-rheumatic drug(DMARD). In one example, the DMARD is selected from the group consistingof hydroxycloroquine, sulfasalazine, methotrexate, leflunomide,azathioprine, D-penicillamine, gold salts minocycline, cyclosporine andTNF-inhibitors.

In one example, the DMARD is selected from the group consisting ofazathioprine, chloroquine, hydroxychloroquine, leflunomide, methotrexateand sulfasalazine. In one example, the DMARD is methotrexate.

In another example, the DMARD is an anti-TNF antibody (e.g., infliximab,golimumab or adalimumab) or a soluble TNF receptor (e.g., etanercept).

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered together with a Bcell depleting agent. In one example, the B cell depleting agent is ananti-CD20 antibody, such as rituximab or ofatumumab.

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered to a subjectsuffering from rheumatoid arthritis and receiving treatment withmethotrexate.

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered as adjunctive and/orconcomitant therapy to methotrexate therapy.

In one example, the subject suffers from moderately active rheumatoidarthritis or severely active rheumatoid arthritis or moderately toseverely active rheumatoid arthritis.

In one example, the STRO-1⁺ cells and/or progeny cells thereof areadministered to a subject suffering from rheumatoid arthritis andreceiving treatment with methotrexate, wherein the cells areadministered at a dose of about 1×10⁶-about 3×10⁶ cells/kg.

In one example, the STRO-1⁺ cells and/or progeny cells thereof areadministered to a subject suffering from rheumatoid arthritis andreceiving treatment with methotrexate, wherein the cells areadministered at a dose of about 1.5×10⁶-about 2×10⁶ cells/kg.

In one example, the STRO-1⁺ cells and/or progeny cells thereof areadministered to a subject suffering from rheumatoid arthritis andreceiving treatment with methotrexate, wherein the cells areadministered at a dose of about 1.5×10⁶ cells/kg.

In one example, the STRO-1⁺ cells and/or progeny cells thereof areadministered to a subject suffering from rheumatoid arthritis andreceiving treatment with methotrexate, wherein the cells areadministered at a dose of about 2×10⁶ cells/kg.

In one example, the STRO-1⁺ cells and/or progeny cells thereof areadministered to a subject suffering from rheumatoid arthritis andreceiving treatment with methotrexate, wherein the cells areadministered at a dose of between about 100 million cells to about 300million cells irrespective of the weight of the patient, such as,between about 100 million cells to about 200 million cells irrespectiveof the weight of the patient.

In one example, the STRO-1⁺ cells and/or progeny cells thereof areadministered systemically, e.g., intravenously.

Thus, in one example, the present disclosure provides a method fortreating or preventing osteoarthritis in a subject, the methodcomprising intravenously (or systemically) administering to the subjecta population of cells enriched for STRO-1⁺ cells and/or progeny thereofand/or soluble factors derived therefrom. Exemplary cells, dosages andcombination treatments are described herein and are to be taken to applymutatis mutandis to the present example of the disclosure.

In another example, the present disclosure provides a method fortreating or preventing rheumatoid arthritis in a subject, the methodcomprising intravenously (or systemically) administering to the subjecta population of cells enriched for STRO-1⁺ cells and/or progeny thereofand/or soluble factors derived therefrom. Exemplary cells, dosages andcombination treatments are described herein and are to be taken to applymutatis mutandis to the present example of the disclosure.

The present disclosure additionally provides a population of cellsenriched for STRO-1⁺ cells and/or progeny thereof and/or soluble factorsderived therefrom for use in the treatment or prevention of a rheumaticdisease in a subject.

The present disclosure additionally provides for use of a population ofcells enriched for STRO-1⁺ cells and/or progeny thereof and/or solublefactors derived therefrom in the manufacture of a medicament fortreating- or preventing a rheumatic disease in a subject.

Key to Sequence Listing

SEQ ID NO: 1 oligonucleotide for amplifying nucleic acid encoding GAPDH

SEQ ID NO: 2 oligonucleotide for amplifying nucleic acid encoding GAPDH

SEQ ID NO: 3 oligonucleotide for amplifying nucleic acid encoding SDF-1

SEQ ID NO: 4 oligonucleotide for amplifying nucleic acid encoding SDF-1

SEQ ID NO: 5 oligonucleotide for amplifying nucleic acid encoding IL-1β

SEQ ID NO: 6 oligonucleotide for amplifying nucleic acid encoding IL-1β

SEQ ID NO: 7 oligonucleotide for amplifying nucleic acid encoding FLT-1

SEQ ID NO: 8 oligonucleotide for amplifying nucleic acid encoding FLT-1

SEQ ID NO: 9 oligonucleotide for amplifying nucleic acid encoding TNF-α

SEQ ID NO: 10 oligonucleotide for amplifying nucleic acid encoding TNF-α

SEQ ID NO: 11 oligonucleotide for amplifying nucleic acid encoding KDR

SEQ ID NO: 12 oligonucleotide for amplifying nucleic acid encoding KDR

SEQ ID NO: 13 oligonucleotide for amplifying nucleic acid encoding RANKL

SEQ ID NO: 14 oligonucleotide for amplifying nucleic acid encoding RANKL

SEQ ID NO: 15 oligonucleotide for amplifying nucleic acid encodingLeptin

SEQ ID NO: 16 oligonucleotide for amplifying nucleic acid encodingLeptin

SEQ ID NO: 17 oligonucleotide for amplifying nucleic acid encodingCBFA-1

SEQ ID NO: 18 oligonucleotide for amplifying nucleic acid encodingCBFA-1

SEQ ID NO: 19 oligonucleotide for amplifying nucleic acid encodingPPARγ2

SEQ ID NO: 20 oligonucleotide for amplifying nucleic acid encodingPPARγ2

SEQ ID NO: 21 oligonucleotide for amplifying nucleic acid encoding OCN

SEQ ID NO: 22 oligonucleotide for amplifying nucleic acid encoding OCN

SEQ ID NO: 23 oligonucleotide for amplifying nucleic acid encoding MyoD

SEQ ID NO: 24 oligonucleotide for amplifying nucleic acid encoding MyoD

SEQ ID NO: 25 oligonucleotide for amplifying nucleic acid encoding SMMHC

SEQ ID NO: 26 oligonucleotide for amplifying nucleic acid encoding SMMHC

SEQ ID NO: 27 oligonucleotide for amplifying nucleic acid encoding GFAP

SEQ ID NO: 28 oligonucleotide for amplifying nucleic acid encoding GFAP

SEQ ID NO: 29 oligonucleotide for amplifying nucleic acid encodingNestin

SEQ ID NO: 30 oligonucleotide for amplifying nucleic acid encodingNestin

SEQ ID NO: 31 oligonucleotide for amplifying nucleic acid encoding SOX9

SEQ ID NO: 32 oligonucleotide for amplifying nucleic acid encoding SOX9

SEQ ID NO: 33 oligonucleotide for amplifying nucleic acid encodingCollagen type X

SEQ ID NO: 34 oligonucleotide for amplifying nucleic acid encodingCollagen type X

SEQ ID NO: 35 oligonucleotide for amplifying nucleic acid encodingAggrecan

SEQ ID NO: 36 oligonucleotide for amplifying nucleic acid encodingAggrecan

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Co-expression of TNAP (STRO-3) and the Mesenchymal PrecursorCell Marker, STRO-1^(bright) by Adult Human bone marrow morphonuclearcells (BMMNC). Dual-color immunofluorescence and flow cytometry wasperformed by incubation of STRO-1 MACS-selected BMMNC and indirectlylabeled with a goat anti-murine IgM antibody coupled to FITC (x axis),and STRO-3 mAb (murine IgG1) indirectly labeled with a goat anti-murineIgG coupled to PE (y axis). The dot plot histogram represents 5×10⁴events collected as listmode data. The vertical and horizontal lineswere set to the reactivity levels of <1.0% mean fluorescence obtainedwith the isotype-matched control antibodies, 1B5 (IgG) and 1A6.12 (IgM)treated under the same conditions. The results demonstrate that a minorpopulation of STRO-1^(bright) cells co-expressed TNAP (upper rightquadrant) while the remaining STRO-1⁺ cells failed to react with theSTRO-3 mAb.

FIG. 2. Gene expression profile of STRO-1^(bright) or STRO-1^(dim)progeny of cultured and expanded STRO-1^(bright) MPC. Single cellsuspensions of ex vivo expanded bone marrow MPC were prepared bytrypsin/EDTA treatment. Cells were stained with the STRO-1 antibodywhich was subsequently revealed by incubation with goat-anti murineIgM-fluorescein isothiocyanate. Total cellular RNA was prepared frompurified populations of STRO-1^(dim) or STRO-1^(bright) expressingcells, following fluorescence activated cell sorting (A). Using RNAzolBextraction method, and standard procedures, total RNA was isolated fromeach subpopulation and used as a template for cDNA synthesis. Theexpression of various transcripts was assessed by PCR amplification,using a standard protocol as described previously (Gronthos et al. JCell Sci. 116:1827-1835, 2003). Primers sets used in this study areshown in Table 2. Following amplification, each reaction mixture wasanalyzed by 1.5% agarose gel electrophoresis, and visualized by ethidiumbromide staining (B). Relative gene expression for each cell marker wasassessed with reference to the expression of the house-keeping gene,GAPDH, using ImageQant software (C).

FIG. 3. STRO-1^(bright) progeny of cultured and expanded STRO-1⁺ MPCexpress high levels of SDF-1, STRO-1^(dim) progeny do not. (A)MACS-isolated preparations of STRO-1⁺ BMMNCs were partitioned intodifferent STRO-1 subsets according to the regions, STRO-1^(bright) andSTRO-1^(dim/dull) using FACS. Total RNA was prepared from each STRO-1subpopulation and used to construct a STRO-1^(bright) subtractionhybridization library (B-C). Replicate nitrocellulose filters, whichhave been blotted with representative PCR products amplified frombacterial clones transformed with STRO-1^(bright) subtracted cDNA. Thefilters were then probed with either [³²P] deoxycytidine triphosphate(dCTP)-labeled STRO-1^(bright) (B) or STRO-1^(dim/dull) (C) subtractedcDNA. The arrows indicate differential expression of 1 clone containinga cDNA fragment corresponding to human SDF-1. (D) Reverse transcriptase(RT)-PCR analysis demonstrating the relative expression of SDF-1 andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts in totalRNA prepared from freshly MACS/FACS-isolated BMMNC STRO-1 populationsprior to culture. bp indicates base pair.

FIG. 4 is a graphical representation showing lameness score of a sheepmodel of rheumatoid arthritis. Scores were determined as describedherein.

FIG. 5 is a graphical representation showing leukocyte counts in thesynovial fluid from right (unstimulated) and left (stimulated) hocks ofindividual sheep during the course of the trial (as indicated in thedrawing). Sheep B1627 and B4036 were immunised with chicken Type IIcollagen and all other sheep were immunised with bovine Type IIcollagen. In the higher responders the leukocyte concentration in thecontrol side was also raised indicating the possibility of a systemicresponse.

FIG. 6 is a graphical representation showing leukocyte counts in thesynovial fluid of control sheep (n=7) and sheep immunised with bovineType II collagen (n=5). Data shows the mean±standard error.

FIG. 7A is a graphical representation showing levels of IgM (left handpanel) and IgG (right hand panel) antibodies to bovine Type II collagenin the synovial fluid from right (control) and left (stimulated) hocksof 5 sheep immunised with bovine Type II collagen. Synovial fluid wastaken at autopsy, 42 days after initial immunisation. Levels of IgM wereraised 4-8 fold and levels of IgG were raised 10-60 fold in thestimulated hocks.

FIG. 7B is a graphical representation showing levels of IgM (left handpanel) and IgG (right hand panel) antibodies to chicken Type II collagenin the synovial fluid from right (control) and left (stimulated) hocksof 2 sheep immunised with chicken Type II collagen. Synovial fluid wastaken at autopsy, 42 days after initial immunisation. Levels of IgM wereraised 3-5 fold and levels of IgG were raised 2-8×106 fold in thestimulated hocks.

FIG. 8A shows aggregate and individual histopathology scores(hyperplasia, stromal activation, inflammatory cell infiltrate) forsynovial membranes from left hock joints of all groups. Anova=p<0.04; pfrom Mann-Whitney test.

FIG. 8B shows aggregate and individual histopathology for synovialmembranes from right hock joints of all IV injected groups.

FIG. 8C shows aggregate and individual histopathology scores forsynovial membranes from groups injected Intra-articularly (IA) withsaline or MPC.

FIG. 9A shows CD14 scoring system (intima scoring scheme as described inMo et al., J Rheumatol. 38: 2301-2308, 2011).

FIG. 9B shows discrete cell scoring scheme applied to CD4, CD8,Gamma-delta TCR, CD79a and Ki-67.

FIG. 9C shows cytokine and adhesion molecule scoring system applied toVCAM-1, IL-6, IL-10, IL-113, IL-17, TNFα and interstitial CD14.

FIG. 10A shows mean±SD immunohistological scores for CD4, CD8,Gamma-delta TCR and CD79a in left hock joint synovial tissues for thecontrol and MPC injected groups.

FIG. 10B shows mean±SD immunohistological scores for VCAM-intima,VCAM-interstitial, Ki67-intima and Ki67-interstitial in left hock jointsynovial tissues for the control and MPC injected groups.

FIG. 10C shows mean±SD immunohistological scores for IL-10-intima,IL-10-interstitial, IL1beta-intima and IL1beta-interstitial in left hockjoint synovial tissues for the control and MPC injected groups.

FIG. 10D shows mean+SEM for synovial immunohistochemical staining scoresfor IL-6 and TNF-alpha for the synovial intima and interstitial tissuesof the IV saline control and MPC injected groups. The p values shownwere calculated using the Mann-Whitney non-parametric t-test.

FIG. 10E shows mean+SEM for synovial immunohistochemical interstitialtissue staining for CD14 positive cells for saline control and MPCinjected groups. The p values shown were calculated using theMann-Whitney non-parametric t-test.

FIG. 10F shows mean+SEM for synovial immunohistochemical interstitialtissue staining for IL-17 for saline control and MPC injected groups.The p values shown were derived from ANOVA with Krushal-Wallis test &Dunns post-hoc test.

FIG. 11A shows photomicrographs of synovial sections immunostained forCD14 cells showing assigned scores. Magnification×100.

FIG. 11B shows photomicrographs of synovial sections immunostained forTNF-α showing assigned scores. Magnification×100.

FIG. 11C shows photomicrographs of synovial sections showingimmunostained for IL-6 showing assigned scores. Magnification×100.

FIG. 11D shows photomicrographs of synovial sections immunostained forIL-17 showing assigned scores. Magnification×100.

FIG. 11E shows photomicrographs of synovial sections immunostained forIL-10 showing assigned scores. Magnification×100.

DETAILED DESCRIPTION

General Techniques and Selected Definitions

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

Each embodiment or example described herein is to be applied mutatismutandis to each and every other embodiment unless specifically statedotherwise.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

The present invention is performed without undue experimentation using,unless otherwise indicated, conventional techniques of molecularbiology, microbiology, virology, recombinant DNA technology, peptidesynthesis in solution, solid phase peptide synthesis, and immunology.Such procedures are described, for example, in Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, New York, Second Edition (1989), whole of Vols I, II, andIII; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover,ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis:A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole oftext, and particularly the papers therein by Gait, ppl-22; Atkinson etal, pp 35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4.Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J.Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cellsand Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole oftext; Perbal, B., A Practical Guide to Molecular Cloning (1984); MethodsIn Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.),whole of series; J. F. Ramalho Ortigao, “The Chemistry of PeptideSynthesis” In: Knowledge database of Access to Virtual Laboratorywebsite (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E.Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342;Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G.and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer,J. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12. Wünsch, E.,ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der OrganischenChemie (Miller, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme,Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis,Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) ThePractice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky,M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook ofExperimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell,eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture:Practical Approach, Third Edition (John R. W. Masters, ed., 2000), ISBN0199637970, whole of text.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers but not the exclusionof any other step or element or integer or group of elements orintegers.

As used herein the term “derived from” shall be taken to indicate that aspecified integer may be obtained from a particular source albeit notnecessarily directly from that source. In the context of soluble factorsderived from STRO-1⁺ cells and/or progeny cells thereof, this term shallbe taken to mean one or more factors, e.g., proteins, peptides,carbohydrates, etc, produced during in vitro culturing of STRO-1⁺ cellsand/or progeny cells thereof.

As used herein, the term “rheumatic diseases” shall be taken to mean anydisease characterized by an inflammatory response in at least one jointin a subject. In one example, the rheumatic disease is caused by orassociated with an autoimmune condition. Exemplary rheumatic diseasesare known in the art and/or described herein.

As used herein, the term “effective amount” shall be taken to mean asufficient quantity of STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom to reduce inflammation and/or thenumber of inflammatory cells and/or inflammatory cytokines in the jointof a subject that causes or is associated with a rheumatic disease.

As used herein, the term “therapeutically effective amount” shall betaken to mean a sufficient quantity of STRO-1⁺ cells and/or progenycells thereof and/or soluble factors derived therefrom to reduce orinhibit one or more symptoms of a rheumatic disease.

As used herein, the term “prophylactically effective amount” shall betaken to mean a sufficient quantity of STRO-1⁺ cells and/or progenycells thereof and/or soluble factors derived therefrom to prevent orinhibit or delay the onset of one or more detectable symptoms of aninflammatory joint disease.

As used herein, the term “low dose” shall be understood to mean anamount of STRO-1⁺ cells and/or progeny thereof less than 1×10⁶ cells/kg,yet still sufficient to reduce inflammation and/or an inflammatorycytokine and/or the number of inflammatory cells in the joint of asubject. For example, a low dose comprises 0.5×10⁶ or fewer cells, or0.4×10⁶ or fewer cells or 0.3×10⁶ or fewer cells or 0.1×10⁶ or fewercells per kg.

As used herein, the term “high dose” shall be understood to mean anamount of STRO-1⁺ cells and/or progeny thereof sufficient to reduceIL-6, TNFα, IL-17 in addition to CD14⁺ cells in a subject, e.g., withina joint of a subject, such as within synovial tissue, such as more than1.5×10⁶ cells/kg. For example, a dose comprises between about 1.5×10⁶ toabout 4×10⁶ cells/kg. For example, a high dose comprises about 1.5×10⁶or about 2×10⁶/kg.

As used herein, the term “treat” or “treatment” or “treating” shall beunderstood to mean administering a therapeutically effective amount ofsoluble factors and/or cells and reducing or inhibiting at least onesymptom of an inflammatory joint, disease.

As used herein, the term “prevent” or “preventing” or “prevention” shallbe taken to mean administering a prophylactically effective amount ofsoluble factors and/or cells and stopping or hindering or delaying thedevelopment or progression of an inflammatory joint disease.

As used herein, the term “soluble factors” shall be taken to mean anymolecule, e.g., protein; peptide, glycoprotein, glycopeptide,lipoprotein, lipopeptide, carbohydrate, etc. produced by STRO-1⁺ cellsand/or progeny thereof that are water soluble. Such soluble factors maybe intracellular and/or secreted by a cell. Such soluble factors may bea complex mixture (e.g., supernatant) and/or a fraction thereof and/ormay be a purified factor. In one example of the present inventionsoluble factors are or are contained within supernatant. Accordingly,any example herein directed to administration of one or more solublefactors shall be taken to apply mutatis mutandis to the administrationof supernatant.

As used herein, the term “supernatant” refers to the non-cellularmaterial produced following the in vitro culturing of mesenchymalprecursor cells, and/or progeny cells thereof, in a suitable medium,such as, liquid medium. Typically, the supernatant is produced byculturing the cells in the medium under suitable conditions and time,followed by removing the cellular material by a process such ascentrifugation. The supernatant may or may not have been subjected tofurther purification steps before administration. In one example, thesupernatant comprises less than 10⁵, such as less than 10⁴, for example,less than 10³ and e.g., no live cells.

As used herein, the term “normal or healthy subject” shall be taken tomean a subject that does not suffer from an inflammatory joint diseaseas assessed by any method known in the art and/or described herein.

As used herein, the term “subject” shall be taken to mean any animalincluding humans, for example a mammal. Exemplary subjects include butare not limited to humans, primates, livestock (e.g. sheep, cows,horses, donkeys, pigs), companion animals (e.g. dogs, cats), laboratorytest animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captivewild animals (e.g. fox, deer). In one example, the mammal is a human orprimate. In one example, the mammal is a human. In one example, asubject is eligible for treatment who is experiencing or has experiencedone or more signs, symptoms, or, other indicators of inflammatory jointdamage, has been diagnosed with inflammatory joint damage, whether, forexample, newly diagnosed or previously diagnosed and now experiencing arecurrence or relapse, or is at risk for developing inflammatory jointdamage.

Rheumatic Diseases

In one example of the present disclosure, rheumatic diseases areinflammatory joint diseases. Inflammatory joint diseases is used hereinin the broadest sense and refers to damage or partial or completedestruction to any part of one or more joints, including the connectivetissue and cartilage, where damage includes structural and/or functionaldamage of any cause and is characterized by inflammation in the joint,and may or may not cause joint pain/arthalgia. This damage may be causedby any condition, such as an autoimmune disease such as arthritis (e.g.,acute and chronic arthritis), rheumatoid arthritis includingjuvenile-onset rheumatoid arthritis, juvenile idiopathic arthritis (HA),or juvenile RA (JRA), and stages such as rheumatoid synovitis, gout orgouty arthritis, acute immunological arthritis, chronic inflammatoryarthritis, degenerative arthritis, type II collagen-induced arthritis,infectious arthritis, septic arthritis, Lyme arthritis, proliferativearthritis, psoriatic arthritis, Still's disease, vertebral arthritis,osteoarthritis, arthritis chronica progrediente, arthritis deformans,polyarthritis chronica primaria, reactive arthritis, menopausalarthritis, estrogen-depletion arthritis, and ankylosingspondylitis/rheumatoid spondylitis), rheumatic autoimmune disease otherthan RA, significant systemic involvement secondary to RA (including butnot limited to vasculitis, pulmonary fibrosis or Felty's syndrome),seronegative spondyloarthropathy, Lyme disease, mixed connective tissuedisease, autoimmune disorders associated with collagen disease.

In one example, the skilled person will understand that an inflammatoryjoint disease is not an injury to cartilage or bone or joint causedsolely by, for example, overuse or a sporting injury or impact to ajoint, since these conditions are not diseases.

In one example, the inflammatory joint disease is associated with orcaused by an autoimmune disease. An “autoimmune disease” is a diseasearising from and directed against a subject's own tissues or organs or aco-segregate or manifestation thereof or resulting condition therefrom.In one example, the inflammatory joint disease is an autoimmuneinflammatory joint disease, such as caused by a subject's having animmune response against an antigen that occurs in the subject's joint.

In one example, the inflammatory joint damage is caused by arthritis,such as, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis,or psoriatic arthritis.

In one example, the inflammatory joint disease is rheumatoid arthritis.

In one example, the inflammatory joint disease is osteoarthritisarthritis.

For purposes herein, joints are points of contact between elements of askeleton (of a vertebrate such as an animal) with the parts thatsurround and support it and include, but are not limited to, forexample, hips, joints between, the vertebrae of the spine, jointsbetween the spine and pelvis (sacroiliac joints), joints where thetendons and ligaments attach to bones, joints between the ribs andspine, shoulders, knees, feet, elbows, hands, fingers, ankles and toes,but especially joints in the hands and feet.

Methods for detecting and/or diagnosing inflammatory joint diseaseand/or monitoring efficacy of treatment and/or if additional treatmentis required or recommended will be apparent to the skilled artisan. Forexample, comparing the number of tender and swollen joints betweenbaseline and various time points during treatment is a typical way toassess joint status and response to treatment. In the American Collegeof Rheumatology (ACR) joint count for RA (Felson et al. Arthritis &Rheumatology 38: 727-735, 1995), 68 joints are assessed for tendernessand 66 for swelling (the hip is not assessed for swelling). In theDisease Activity Score (DAS) employed primarily in Europe, either a 44-or 28-joint count is used in RA. In addition to the joint count, the ACRevaluation criteria include the following elements to comprise acomposite score: patient global (on a visual analog scale [VAS]),patient pain, physician global, Health Assessment Questionnaire (HAQ; ameasure of function), and an acute-phase reactant (either C-reactiveprotein or sedimentation rate). An ACR 20 response would constitute a20% improvement in tender and swollen joint count and a 20% improvementof at least 3 of the other 5 elements in the composite criteria. ACR 50and 70 responses represent at least a 50% and 70% improvement of theseelements. The ACR system only represents change, whereas the DAS systemrepresents both current state of disease activity and change. The DASscoring system uses a weighted mathematical formula, derived fromclinical trials in RA. For example, the DAS 28 is0.56(T28)+0.28(SW28)+0.70(Ln ESR)+0.014 GH wherein T represents tenderjoint number, SW is swollen joint number, ESR is erythrocytesedimentation rate, and GH is global health. Various values of the DASrepresent high or low disease activity as well as remission, and thechange and endpoint score result in a categorization of the patient bydegree of response (none, moderate, good).

As used herein, “rheumatoid arthritis” refers to a recognized diseasestate which may be diagnosed according to the 2000 revised AmericanRheumatoid Association criteria for the classification of rheumatoidarthritis, or any similar criteria. Physiological indicators of RAinclude, symmetric joint swelling which is characteristic though notinvariable in rheumatoid arthritis. Fusiform swelling of the proximalinterphalangeal (PIP) joints of the hands as well as metacarpophalangeal(MCP), wrists, elbows, knees, ankles and metatarsophalangeal (MTP)joints are commonly affected and swelling is easily detected. Pain onpassive motion is the most sensitive test for joint inflammation, andinflammation and structural deformity often limits the range of motionfor the affected joint. Typical visible changes include ulnar deviationof the fingers at the MCP joints, hyperextension or hyperflexion of theMCP and PIP joints, flexion contractures of the elbows, and subluxationof the carpal bones and toes. The subject with rheumatoid arthritis mayor may not be resistant to DMARDs, in that the DMARDs are not effectiveor fully effective in treating symptoms. Furthermore, the subject mayhave experienced an inadequate response to previous or current treatmentwith TNF inhibitors such as etanercept, infliximab and/or adalimumabbecause of toxicity or inadequate efficacy (for example, etanercept for3 months at 25 mg twice a week or at least 4 infusions of infliximab at3 mg/kg) and/or anti-CD20 therapy (e.g., rituximab.

Rheumatoid arthritis can also be diagnosed by the presence ofautoantibodies, e.g., rheumatoid factor (antibodies that bind IgG)and/or anti-cyclic citrullinated peptide and/or heterogeneous nuclearribonucleoprotein A2 (RA33) and/or type II collagen and/or stressproteins (e.g., BiP or hsp90) and/or glucose 6-phophate isomerise (GPI).

“Psoriatic arthritis” or “PsA” is a chronic disease characterized byinflammation of the skin (psoriasis) and joints (arthritis). Psoriasisfeatures patchy, raised, red areas of skin inflammation with scaling andoften affects the tips of the elbows and knees, the scalp, the navel,and around the genital areas or anus. Approximately 10% of patients whohave psoriasis also develop an associated inflammation of their joints.Patients who have both inflammatory arthritis and psoriasis arediagnosed as having psoriatic arthritis. Psoriatic arthritis is asystemic rheumatic disease that can also cause inflammation in bodytissues away from the joints and the skin, such as in the eyes, heart,lungs, and kidneys.

“Ankylosing spondylitis” or “AS” is a form of chronic inflammation ofthe spine and the sacroiliac joints, which are located in the low backwhere the sacrum (the bone directly above the tailbone) meets the iliacbones (bones on either side of the upper buttocks). Chronic inflammationin these areas causes pain and stiffness in and around the spine. Overtime, chronic spinal inflammation (spondylitis) can lead to a completecementing together (fusion) of the vertebrae, a process referred to asankylosis. Ankylosis leads to loss of mobility of the spine. Ankylosingspondylitis is also a systemic rheumatic disease, meaning it can affectother tissues throughout the body. Accordingly, it can causeinflammation in or injury to other joints away from the spine, as wellas other organs, such as the eyes, heart, lungs, and kidneys.

MPCs or Progeny Cells, and Supernatant or One or More Soluble FactorsDerived Therefrom

MPCs are cells found in bone marrow, blood, dental pulp cells, adiposetissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina,brain, hair follicles, intestine, lung, lymph node, thymus, bone,ligament, tendon, skeletal muscle, dermis, and periosteum; and arecapable of differentiating into germ lines such as mesoderm and/orendoderm and/or ectoderm.

In one example, the MPCs are multipotential cells which are capable ofdifferentiating into a large number of cell types including, but notlimited to, adipose, osseous, cartilaginous, elastic, muscular, andfibrous connective tissues. The specific lineage-commitment anddifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironinental conditions established by host tissues. MPCs aremultipotential cells are thus non-hematopoietic progenitor cells whichdivide to yield daughter cells that are either stem cells or areprecursor cells which in time will irreversibly differentiate to yield aphenotypic cell.

MPCs are positive for the marker STRO-1 (i.e. MPCs are STRO-1⁺ cells)

In one example, the STRO-1⁺ cells are enriched from a sample obtainedfrom a subject, e.g., a subject to be treated or a related subject or anunrelated subject (whether of the same species or different). The terms“enriched”, “enrichment” or variations thereof are used herein todescribe a population of cells in which the proportion of one particularcell type or the proportion of a number of particular cell types isincreased when compared with an untreated population of the cells (e.g.,cells in their native environment). In one example, a populationenriched for STRO-1⁺ cells comprises at least about 0.1% or 0.5% or 1%or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75%STRO-1^(+ cells. In this regard, the term “population of cells enriched for STRO-)1⁺cells” will be taken to provide explicit support for the term“population of cells comprising X % STRO1⁺ cells”, wherein X % is apercentage as recited herein. The STRO-1⁺ cells can, in some examples,form clonogenic colonies, e.g. CFU-F (fibroblasts) or a subset thereof(e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

In one example, the population of cells is enriched from a cellpreparation comprising STRO-1⁺ cells in a selectable form. In thisregard, the term “selectable form” will be understood to mean that thecells express a marker (e.g., a cell surface marker) permittingselection of the STRO-1⁺ cells. The marker can be STRO-1, but need notbe. For example, as described and/or exemplified herein, cells (e.g.,MPCs) expressing STRO-2 and/or STRO-3 (TNAP) and/or STRO-4 and/or VCAM-1and/or CD146 and/or 3G5 also express STRO-1 (and can beSTRO-1^(bright)). Accordingly, an indication that cells are STRO-1⁺ doesnot necessarily mean that the cells are selected by STRO-1 expression.In one example, the cells are selected based on at least STRO-3expression, e.g., they are STRO-3⁺ (TNAP⁺).

Reference to selection of a cell or population thereof does not requireselection from a specific tissue source. As described herein STRO-1⁺cells can be selected from or isolated from or enriched from a largevariety of sources. That said, in some examples, these terms providesupport for selection from any tissue comprising STRO-1⁺ cells (e.g.,MPCs) or vascularized tissue or tissue comprising pericytes (e.g.,STRO-1⁺ pericytes) or any one or more of the tissues recited herein.

In one example, the cells used in the present invention express one ormore markers individually or collectively selected from the groupconsisting of TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺, 3G5⁺ orany combination thereof.

By “individually” is meant that the invention encompasses the recitedmarkers or groups of markers separately, and that, notwithstanding thatindividual markers or groups of markers may not be separately listedherein the accompanying claims may define such marker or groups ofmarkers separately and divisibly from each other.

By “collectively” is meant that the invention encompasses any number orcombination of the recited markers or groups of peptides, and that,notwithstanding that such numbers or combinations of markers or groupsof markers may not be specifically listed herein the accompanying claimsmay define such combinations or sub-combinations separately anddivisibly from any other combination of markers or groups of markers.

For example, the STRO-1⁺ cells are STRO-1^(bright) (syn. STRO-1^(bri)).In one example, the Stro-1^(bri) cells are preferentially enrichedrelative to STRO-1^(dim) or STRO-1^(intermediate) cells.

For example, the STRO-1^(bright) cells are additionally one or more ofTNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺ and/or CD146⁺. For example, the cellsare Selected for one or more of the foregoing markers and/or shown toexpress one or more of the foregoing markers. In this regard, a cellshown to express a marker need not be specifically tested, ratherpreviously enriched or isolated cells can be tested and subsequentlyused, isolated or enriched cells can be reasonably assumed to alsoexpress the same marker.

In one example, the mesenchymal precursor cells are perivascularmesenchymal precursor cells as defined in WO 2004/85630. For example,the mesenchymal precursor cells express a marker of a perivascular cell,e.g., the cells are STRO-1⁺ or STRO-1^(bright) and/or 3G5⁺. In oneexample, the cells are or were previously or are progeny of cells thatwere isolated from vascularized tissue or organs or parts thereof.

A cell that is referred to as being “positive” for a given marker it mayexpress either a low (lo or dim) or a high (bright, bri) level of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother marker used in the sorting process of the cells. The distinctionof lo (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being sorted. A cell that isreferred to as being “negative” for a given marker is not necessarilycompletely absent from that cell. This term means that the marker isexpressed at a relatively very low level by that cell, and that itgenerates a very low signal when detectably labeled or is undetectableabove background levels, e.g., levels detected suing an isotype controlantibody.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectably labeled.Whilst not wishing to be limited by theory, it is proposed that “bright”cells express more of the target marker protein (for example the antigenrecognized by STRO-1) than other cells in the sample. For instance,STRO-1^(bri) cells produce a greater fluorescent signal, when labeledwith a FITC-conjugated STRO-1 antibody as determined by fluorescenceactivated cell sorting (FACS) analysis, than non-bright cells(STRO-1^(dull/dim)). For example, “bright” cells constitute at leastabout 0.1% of the most brightly labeled bone marrow mononuclear cellscontained in the starting sample. In other examples, “bright” cellsconstitute at least about 0.1%, at least about 0.5%, at least about 1%,at least about 1.5%, or at least about 2%, of the most brightly labeledbone marrow mononuclear cells contained in the starting sample. In oneexample, STRO-1^(bri)g^(ht) cells have 2 log magnitude higher expressionof STRO-1 surface expression relative to “background”, namely cells thatare STRO-1⁻. By comparison, STRO-1^(dim) and/or STRO-1^(intermediate)cells have less than 2 log magnitude higher expression of STRO-1 surfaceexpression, typically about 1 log or less than “background”.

As used herein the term “TNAP” is intended to encompass all isoforms oftissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In one example, the TNAP is BAP. In one example,TNAP as used herein refers to a molecule which can bind the STRO-3antibody produced by the hybridoma cell line deposited with ATCC on 19Dec. 2005 under the provisions of the Budapest Treaty under depositaccession number PTA-7282.

Furthermore, in one example, the STRO-1⁺ cells are capable of givingrise to clonogenic CFU-F.

In one example, a significant proportion of the STRO-1⁺ multipotentialcells are capable of differentiation into at least two different germlines. Non-limiting examples of the lineages to which the multipotentialcells may be committed include bone precursor cells; hepatocyteprogenitors, which are multipotent for bile duct epithelial cells andhepatocytes; neural restricted cells, which can generate glial cellprecursors that progress to oligodendrocytes and astrocytes; neuronalprecursors that progress to neurons; precursors for cardiac muscle andcardiomyocytes, glucose-responsive insulin secreting pancreatic betacell lines. Other lineages include, but are not limited to,odontoblasts, dentin-producing cells and chondrocytes, and precursorcells of the following: retinal pigment epithelial cells, fibroblasts,skin cells such as keratinocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal muscle, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells.

In another example, the STRO-1⁺ cells are not capable of giving rise,upon culturing, to hematopoietic cells.

In one example, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative example, cells of one or more of the established human celllines are used. In another useful example of the invention, cells of anon-human animal (or if the patient is not a human, from anotherspecies) are used.

The present invention also contemplates use of supernatant or solublefactors obtained or derived from STRO-1⁺ cells and/or progeny cellsthereof (the latter also being referred, to as expanded cells) which areproduced from in vitro culture. Expanded cells of the invention may ahave a wide variety of phenotypes depending on the culture conditions(including the number and/or type of stimulatory factors in the culturemedium), the number of passages and the like. In certain examples, theprogeny cells are obtained after about 2, about 3, about 4, about 5,about 6, about 7; about 8, about 9, or about 10 passages from theparental population. However, the progeny cells may be obtained afterany number of passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. A powder mixture that whenmixed with water or other liquid becomes suitable for cell culture maybe termed a “powdered medium”.

In an example, progeny cells useful for the methods of the invention areobtained by isolating TNAP⁺ STRO-1⁺ cells from bone marrow usingmagnetic beads labeled with the STRO-3 antibody, and then cultureexpanding the isolated cells (see Gronthos et al. Blood 85: 929-940,1995 for an example of suitable culturing conditions).

In one example, such expanded cells (progeny) (for example, at leastafter 5 passages) can be TNAP⁻, CC9⁺, HLA class I⁺, HLA class II⁻,CD14⁻, CD19⁻, CD3⁻, CD1a⁻c⁻, CD31⁻, CD86⁻, CD34⁻ and/or CD80⁻. However,it is possible that under different culturing conditions to thosedescribed herein that the expression of different markers may vary.Also, whilst cells of these phenotypes may predominate in the expendedcell population it does not mean that there is a minor proportion of thecells do not have this phenotype(s) (for example, a small percentage ofthe expanded cells may be CC9⁻). In one example, expanded cells stillhave the capacity to differentiate into different cell types.

In one example, an expended cell population used to obtain supernatantor soluble factors, or cells per se, comprises cells wherein at least25%, for example at least 50%, of the cells are CC9⁺.

In another example, an expanded cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, for example at least 45%, of the cells are STRO-1⁺.

In a further example, the expanded cells may express one or more markerscollectively or individually selected from the group consisting ofLFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5,CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61,integrin beta 6-19, thrombomodulin, CD 10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, Leptin-R (STRO-2=Leptin-R), RANKL, STRO-1^(bright)and CD146 or any combination of these markers.

In one example, the progeny cells are Multipotential Expanded STRO-1⁺Multipotential cells Progeny (MEMPs) as defined and/or described in WO2006/032092. Methods for preparing enriched populations of STRO-1⁺multipotential cells from which progeny may be derived are described inWO 01/04268 and WO 2004/085630. In an in vitro context STRO-1⁺multipotential cells will rarely be present as an absolutely purepreparation and will generally be present with other cells that aretissue specific committed cells (TSCCs). WO 01/04268 refers toharvesting such cells from bone marrow at purity levels of about 0.1% to90%. The population comprising MPCs from which progeny are derived maybe directly harvested from a tissue source, or alternatively it may be apopulation that has already been expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified STRO-1⁺ multipotential cells,comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or95% of total cells of the population in which they are present. Thislevel may be achieved, for example, by selecting for cells that arepositive for at least one marker individually or collectively selectedfrom the group consisting of TNAP, STRO-1^(bright), 3G5⁺, VCAM-1, THY-1,CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested STRO-1⁺ multipotentialcells in that they are positive for the marker STRO-1^(bri) and negativefor the marker Alkaline phosphatase (ALP). In contrast, freshly isolatedSTRO-1⁺ multipotential cells are positive for both STRO-1^(bri) and ALP.In one example of the present invention, at least 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 95% of the administered cells have thephenotype STRO-1^(bri), ALP⁻. In a further example the MEMPS arepositive for one or more of the markers Ki67, CD44 and/or CD49c/CD29,VLA-3, α3β1. In yet a further example the MEMPs do not exhibit TERTactivity and/or are negative for the marker CD18.

The STRO-1⁺ cell starting population may be derived from any one or moretissue types set out in WO 01/04268 or WO 2004/085630, namely bonemarrow, dental pulp cells, adipose tissue and skin, or perhaps morebroadly from adipose tissue, teeth, dental pulp, skin, liver, kidney,heart, retina, brain, hair follicles, intestine, lung, spleen, lymphnode, thymus, pancreas, bone, ligament, bone marrow, tendon and skeletalmuscle.

It will be understood that in performing the present invention,separation of cells carrying any given cell surface marker can beeffected by a number of different methods, however, exemplary methodsrely upon binding a binding agent (e.g., an antibody or antigen bindingfragment thereof) to the marker concerned followed by a separation ofthose that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody-based molecules, with exemplary methods beingmonoclonal antibodies or based on monoclonal antibodies because of thespecificity of these latter agents. Antibodies can be used for bothsteps, however other agents might also be used, thus ligands for thesemarkers may also be employed to enrich for cells carrying them, orlacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. Exemplary separation techniques maximize theretention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS. Methods for performing FACS will be apparent to the skilledartisan.

Antibodies against each of the markers described herein are commerciallyavailable (e.g., monoclonal antibodies against STRO-1 are commerciallyavailable from R&D Systems, USA), available from ATCC or otherdepositary organization and/or can be produced using art recognizedtechniques:

Exemplary methods for isolating STRO-1⁺ cells comprises a first stepbeing a solid phase sorting step utilizing for example magneticactivated cell sorting (MACS) recognizing high level expression ofSTRO-1. A second sorting step can then follow, should that be desired,to result in a higher level of precursor cell expression as described inpatent specification WO 01/14268. This second sorting step might involvethe use of two or more markers.

The method obtaining STRO-1⁺ cells might also include the harvesting ofa source of the cells before the first enrichment step using knowntechniques. Thus the tissue will be surgically removed. Cells comprisingthe source tissue will then be separated into a so called single cellssuspension. This separation may be achieved by physical and or enzymaticmeans.

Once a suitable STRO-1⁺ cell population has been obtained, it may becultured or expanded by any suitable means to obtain MEMPs.

In one example, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative example, cells of one or more of the established human celllines are used to obtain the supernatant or soluble factors. In anotheruseful example of the invention, cells of a non-human animal (or if thepatient is not a human, from another species) are used to obtainsupernatant or soluble factors.

The invention can be practiced using cells from any non-human animalspecies, including but not limited to non-human primate cells, ungulate,canine, feline, lagomorph, rodent, avian, and fish cells. Primate cellswith which the invention may be performed include but are not limited tocells of chimpanzees, baboons, cynomolgus monkeys, and any other New orOld World monkeys. Ungulate cells with which the invention may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Examples of lagomorph specieswith which the invention may be performed include domesticated rabbits,jack rabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which theinvention may be performed.

Cells useful for the methods of the invention may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Anymethod maintaining the biological activity of the isolated stem cellssuch as mesenchymal stem/progenitor cells, or progeny thereof, may beutilized in connection with the present invention. In one example, thecells are maintained and stored by using cryo-preservation.

Genetically-Modified Cells

In one example, the STRO-1⁺ cells and/or progeny cells thereof aregenetically modified, e.g., to express and/or secrete a protein ofinterest. For example, the cells are engineered to express a proteinuseful in the treatment of an inflammatory joint disease, such as ananti-TNF antibody (e.g., adalimumab or infliximab) or an anti-CD20antibody (e.g., rituximab or ocrelizumab) or a soluble TNF receptor(e.g., etanercept) or a peptide useful for treating such conditions(e.g., as described U.S. Pat. No. 5,837,686).

Methods for genetically modifying a cell will be apparent to the skilledartisan. For example, a nucleic acid that is to be expressed in a cellis operably-linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter operable in avariety of cells of a subject, such as, for example, a viral promoter,e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter.Additional suitable promoters are known in the art and shall be taken toapply mutatis mutandis to the present example of the invention.

For example, the nucleic acid is provided in the form of an expressionconstruct. As used herein, the term “expression construct” refers to anucleic acid that has the ability to confer expression on a nucleic acid(e.g. a reporter gene and/or a counter-selectable reporter gene) towhich it is operably connected, in a cell. Within the context of thepresent invention, it is to be understood that an expression constructmay comprise or be a plasmid, bacteriophage, phagemid, cosmid, virussub-genomic or genomic fragment, or other nucleic acid capable ofmaintaining and/or replicating heterologous DNA in an expressibleformat.

Methods for the construction of a suitable expression construct forperformance of the invention will be apparent to the skilled artisan andare described, for example, in Ausubel et al (In: Current Protocols inMolecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).For example, each of the components of the expression construct isamplified from a suitable template nucleic acid using, for example, PCRand subsequently cloned into a suitable expression construct, such asfor example, a plasmid or a phagemid.

Vectors suitable for such an expression construct are known in the artand/or described herein. For example, an expression vector suitable forthe method of the present invention in a mammalian cell is, for example,a vector of the pcDNA vector suite supplied by Invitrogen, a vector ofthe pCI vector suite (Promega), a vector of the pCMV vector suite(Clontech), a pM vector (Clontech), a pSI vector (Promega), a VP 16vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen).

The skilled artisan will be aware of additional vectors and sources ofsuch vectors, such as, for example, Life Technologies Corporation,Clontech or Promega.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given organism depends onthe known successful techniques. Means for introducing recombinant DNAinto cells include microinjection, transfection mediated byDEAE-dextran, transfection mediated by liposomes such as by usinglipofectamine (Gibco, Md., USA) and/or cellfectin (Gibco, Md., USA),PEG-mediated DNA uptake, electroporation and microparticle bombardmentsuch as by using DNA-coated tungsten or gold particles (Agracetus Inc.,WI, USA) amongst others.

Alternatively, an expression construct of the invention is a viralvector. Suitable viral vectors are known in the art and commerciallyavailable. Conventional viral-based systems for the delivery of anucleic acid and integration of that nucleic acid into a host cellgenome include; for example, a retroviral vector, a lentiviral vector oran adeno-associated viral vector. Alternatively, an adenoviral vector isuseful for introducing a nucleic acid that remains episomal into a hostcell. Viral vectors are an efficient and versatile method of genetransfer in target cells and tissues. Additionally, high transductionefficiencies have been observed in many different cell types and targettissues.

For example, a retroviral vector generally comprises cis-acting longterminal repeats (LTRs) with packaging capacity for up to 6-10 kb offoreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of a vector, which is then used to integratethe expression construct into the target cell to provide long termexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simianimmunodeficiency virus (SrV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J Virol. 56:2731-2739(1992); Johann et al, J. Virol. 65:1635-1640 (1992); Sommerfelt et al,Virol. 76:58-59 (1990); Wilson et al, J. Virol. 63:274-2318 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700; Miller andRosman BioTechniques 7:980-990, 1989; Miller, A. D. Human Gene Therapy7:5-14, 1990; Scarpa et al Virology 75:849-852, 1991; Burns et al. Proc.Natl. Acad. Sci USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for nucleic acid delivery. AAV vectors can be readilyconstructed using techniques known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 andWO 93/03769; Lebkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka.Current Topics in Microbiol, and Immunol. 158:97-129, 1992; Kotin, HumanGene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169,1994; and Zhou et al. J Exp. Med. 179:1867-1875, 1994.

Additional viral vectors useful for delivering an expression constructof the invention include, for example, those derived from the pox familyof viruses, such as vaccinia virus and avian poxvirus or an alphavirusor a conjugate virus vector (e.g. that described in Fisher-Hoch et al.,Proc. Natl Acad. Sci. USA 56:317-321, 1989).

Assaying Therapeutic/Prophylactic Potential of Cells and Soluble Factors

Methods for determining the ability of cells or soluble factors to treator prevent or delay the onset or progression of an inflammatory jointdisease will be apparent to the skilled artisan.

For example, an in vitro model of rheumatoid arthritis is described inSchultz et al., Arthritis and Rheumatism, 40: 1420-1428, 1997 andinvolves culturing synovial membranes and articular cartilage explantsor synovial cells and chondrocytes from subjects suffering fromarthritis in a 3-dimensional fibrin matrix. Administration of cells andor soluble factors described herein to the culture permits determiningthe therapeutic/prophylactic efficacy of the cells/factors, e.g., byassessing expression of proteolytic enzymes, chondrocyte matrixarchitecture, matrix degradation or cell numbers.

Another in vitro model of arthritis involves culturing cartilage discswith synovial fibroblasts in the presence of TNF-alpha and/or IL-1 beta.Administration of cells and or soluble factors described herein to theculture permits determining the therapeutic/prophylactic efficacy of thecells/factors, e.g., by assessing expression of proteolytic enzymes,collagen matrix architecture, matrix degradation, pro-inflammatorycytokine levels (e.g., IL-6 and/or IL-8) or cell numbers.

In another example, efficacy of cells and/or soluble factors describedherein is assessed in an in vivo model of rheumatoid arthritis, e.g., aSKG strain of mouse (Sakaguchi et al., Nature, 426: 454-460), rat typeII collagen arthritis model, mouse type II collagen arthritis model orantigen induced arthritis models in several species (Bendele JMusculoskel Neuron Interact 2001; 1(4):377-385). The inventors have alsodemonstrated a sheep model of rheumatoid arthritis.

Animal models of ankylosing spondylitis are also known in the art, andinclude models involving immunizing balb/c mice with aggrecan and/orversican, ank/ank mice rats overexpressing leukocyte antigen-B27.

It will be apparent to the skilled artisan from the foregoing that thepresent disclosure also provides a method for identifying or isolating acell or a soluble factor for the treatment, prevention or delay of aninflammatory joint disease thereof, the method comprising:

(i) administering a cell or a soluble factor to a test subject sufferingfrom an inflammatory joint disease and assessing inflammation in a jointof the subject;

(ii) comparing level of inflammation in the joint of the subject at (i)to the level of inflammation in the joint of a control subject sufferingfrom the inflammatory joint disease to which the cell or soluble factorhas not been administered,

wherein reduced inflammation in the joint of test subject compared tothe control subject indicates that the cell or soluble factor treats,prevents or delays an inflammatory joint disease.

The present disclosure also provides a method for identifying orisolating a cell or a soluble factor for the treatment, prevention ordelay of an inflammatory joint disease thereof, the method comprising:

(i) contacting a test in vitro model of inflammatory joint disease anddetermining the level of one or more markers of inflammation in themodel;

(ii) determining the level of one or more markers of inflammation in acontrol in vitro model of inflammatory joint disease to which the cellor soluble factor has not been administered,

wherein a reduced level of the marker of inflammation in the test modelcompared to the control model indicates that the cell or soluble factortreats, prevents or delays an inflammatory joint disease.

Exemplary markers of inflammation include expression of proteolyticenzymes, collagen matrix architecture, matrix degradation,pro-inflammatory cytokine levels (e.g., IL-6 and/or IL-8) orinflammatory cell numbers.

The cell may be any cell described herein according to any example.

Cellular Compositions

In one example of the present disclosure STRO-1⁺ cells and/or progenycells thereof are administered in the form of a composition. Forexample, such a composition comprises a pharmaceutically acceptablecarrier and/or excipient.

The terms “carrier” and “excipient” refer to compositions of matter thatare conventionally used in the art to facilitate the storage,administration, and/or the biological activity of an active compound(see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., MacPublishing Company (1980). A carrier may also reduce any undesirableside effects of the active compound. A suitable carrier is, for example,stable, e.g., incapable of reacting with other ingredients in thecarrier. In one example, the carrier does not produce significant localor systemic adverse effect in recipients at the dosages andconcentrations employed for treatment.

Suitable carriers for the present disclosure include thoseconventionally used, e.g., water, saline, aqueous dextrose, lactose,Ringer's solution, a buffered solution, hyaluronan and glycols areexemplary liquid carriers, particularly (when isotonic) for solutions.Suitable pharmaceutical carriers and excipients include starch,cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, magnesium stearate, sodium stearate, glycerol monostearate,sodium chloride, glycerol, propylene glycol, water, ethanol, and thelike.

In another example, a carrier is a media composition, e.g., in which acell is grown or suspended. For example, such a media composition doesnot induce any adverse effects in a subject to whom it is administered.

Exemplary carriers and excipients do not adversely affect the viabilityof a cell and/or the ability of a cell to reduce, prevent or delayinflammatory joint disease.

In one example, the carrier or excipient provides a buffering activityto maintain the cells and/or soluble factors at a suitable pH to therebyexert a biological activity, e.g., the carrier or excipient is phosphatebuffered saline (PBS). PBS represents an attractive carrier or excipientbecause it interacts with cells and factors minimally and permits rapidrelease of the cells and factors, in such a case, the composition of theinvention may be produced as a liquid for direct application to theblood stream or into a tissue or a region surrounding or adjacent to atissue, e.g., by injection.

STRO-1⁺ cells and/or progeny cells thereof can also be incorporated orembedded within scaffolds that are recipient-compatible and whichdegrade into products that are not harmful to the recipient. Thesescaffolds provide support and protection for cells that are to betransplanted into the recipient subjects. Natural and/or syntheticbiodegradable scaffolds are examples of such scaffolds.

A variety of different scaffolds may be used successfully in thepractice of the invention. Exemplary scaffolds include, but are notlimited to biological, degradable scaffolds. Natural biodegradablescaffolds include collagen, fibronectin, and laminin scaffolds. Suitablesynthetic material for a cell transplantation scaffold should be able tosupport extensive cell growth and cell function. Such scaffolds may alsobe resorbable. Suitable scaffolds include polyglycolic acid scaffolds,e.g., as described by Vacanti, et al. J. Ped. Surg. 23:3-9 1988; Cima,et al. Biotechnol. Bioeng. 38:145 1991; Vacanti, et al. Plast. Reconstr.Surg. 88:753-9 1991; or synthetic polymers such as polyanhydrides,polyorthoesters, and polylactic acid.

In another example, the cells may be administered in a gel scaffold(such as Gelfoam from Upjohn Company.

The cellular compositions useful for methods described herein may beadministered alone or as admixtures with other cells. Cells that may beadministered in conjunction with the compositions of the presentinvention include, but are not limited to, other multipotent orpluripotent cells or stem cells, or bone marrow cells. The cells ofdifferent types may be admixed with a composition of the inventionimmediately or shortly prior to administration, or they may beco-cultured together for a period of time prior to administration.

In one example, the composition comprises an effective amount or atherapeutically or prophylactically effective amount of cells. Forexample, the composition comprises about 1×10⁵ STRO-1⁺ cells/kg to about1×10⁷ STRO-1⁺ cells/kg or about 1×10⁶ STRO-1⁺ cells/kg to about 5×10⁶STRO-1⁺ cells/kg. The exact amount of cells to be administered isdependent upon a variety of factors, including the age, weight, and sexof the patient, and the extent and severity of the inflammatory jointdisease

In one example, a low dose of cells is administered to the subject.Exemplary dosages include between about 0.1×10⁴ to about 0.5×10⁶ cellsper kg, for example, between about 0.1×10⁵ to about 0.5×10⁶ cells perkg, such as, between about 0.5×10⁵ to about 0.5×10⁶ cells per kg, forexample, between about 0.1×10⁶ to about 0.5×10⁶ cells per kg, e.g.,about 0.2×10⁶ or 0.3×10⁶ or 0.4×10⁶ cells per kg.

In one example, a high dose of cells is administered to the subject.Exemplary dosages include at least about 1.5×10⁶ cells/kg. For example,a high dose comprises between about 1.5×10⁶ to about 6×10⁶ cells/kg,such as between about 1.5×10⁶ to about 5×10⁶ cells/kg, for example,between about 1.5×10⁶ to about 4×10⁶ cells/kg, for example, betweenabout 1.5×10⁶ to about 3×10⁶ cells/kg. For example, a high dosecomprises about 1.5×10⁶ or about 2×10⁶ cells/kg.

In one example, the cells are administered as a total cell number doseirrespective of the patient's weight.

For example, in one example, the cells are administered at a dose ofbetween about 100 million to 300 million cells irrespective of theweight of the patient.

For example, in one example, the cells are administered at a dose ofbetween about 100 million to 200 million cells irrespective of theweight of the patient.

In one example, the cells are administered at a dose of about 100million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 150million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 200million cells irrespective of the weight of the patient.

In one example, the cells are administered at a dose of about 300million cells irrespective of the weight of the patient.

In some examples, cells are contained within a chamber that does notpermit the cells to exit into a subject's circulation, however thatpermits factors secreted by the cells to enter the circulation. In thismanner soluble factors may be administered to a subject by permittingthe cells to secrete the factors into the subject's circulation. Such achamber may equally be implanted at a site in a subject to increaselocal levels of the soluble factors, e.g., implanted in or near apancreas.

In some examples of the invention, it may not be necessary or desirableto immunosuppress a patient prior to initiation of therapy with cellularcompositions. Accordingly, transplantation with allogeneic, or evenxenogeneic, STRO-1⁺ cells or progeny thereof may be tolerated in someinstances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy and/or reduce an immune response of a subject against thecellular composition. This may be accomplished through the use ofsystemic or local immunosuppressive agents, or it may be accomplished bydelivering the cells in an encapsulated device. The cells may beencapsulated in a capsule that is permeable to nutrients and oxygenrequired by the cell and therapeutic factors the cell is yet impermeableto immune humoral factors and cells. For example, the encapsulant ishypoallergenic, is easily and stably situated in a target tissue, andprovides added protection to the implanted structure. These and othermeans for reducing or eliminating an immune response to the transplantedcells are known in the art. As an alternative, the cells may begenetically modified to reduce their immunogenicity.

Compositions of Soluble Factors

In one example of the present invention, STRO-1⁺ cell-derived and/orprogeny cell-derived supernatant or soluble factors are administered inthe form of a composition, e.g., comprising a suitable carrier and/orexcipient. For example, the carrier or excipient does not adverselyaffect the biological effect of the soluble factors or supernatant.

In one example, the composition comprises a composition of matter tostabilize a soluble factor or a component of supernatant, e.g., aprotease inhibitor. For example, the protease inhibitor is not includedin an amount sufficient to have an adverse effect on a subject.

Compositions comprising STRO-1⁺ cell-derived and/or progeny cell-derivedsupernatant or soluble factors may be prepared as appropriate liquidsuspensions, e.g., in culture medium or in a stable carrier or a buffersolution, e.g., phosphate buffered saline. Suitable carriers aredescribed herein above. In another example, suspensions comprisingSTRO-1⁺ cell-derived and/or progeny cell-derived supernatant or solublefactors are oily suspensions for injection. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil; or synthetic fattyacid esters, such as ethyl oleate or triglycerides; or liposomes.Suspensions to be used for injection may also contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Sterile injectable solutions can be prepared by incorporating thesupernatant or soluble factors in the required amount in an appropriatesolvent with one or a combination of ingredients described above, asrequired, followed by filtered sterilization.

Generally, dispersions are prepared by incorporating the supernatant orsoluble factors into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, exemplary methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. In accordance with an alternative aspect of theinvention, the supernatant or soluble factors may be formulated with oneor more additional compounds that enhance its solubility.

Other exemplary carriers or excipients are described, for example, inHardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis ofTherapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: TheScience and Practice of Pharmacy, Lippincott, Williams, and Wilkins, NewYork, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: DisperseSystems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, N.Y.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, Liposome, or other orderedstructure. The carrier can be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin. Moreover, the soluble factors may be administered ina time release formulation, for example in a composition which includesa slow release polymer. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid and polylactic, polyglycoliccopolymers (PLG). Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art.

The supernatant or soluble factors may be administered in combinationwith an appropriate matrix, for instance, to provide slow release of thesoluble factors.

Additional Components of Compositions

The STRO-1⁺ cell-derived supernatant or soluble factors, STRO-1⁺ cellsor progeny thereof may be administered with other beneficial drugs orbiological molecules (growth factors, trophic factors). Whenadministered with other agents, they may be administered together in asingle pharmaceutical composition, or in separate pharmaceuticalcompositions, simultaneously or sequentially with the other agents(either before or after administration of the other agents). Bioactivefactors which may be co-administered include anti-apoptotic agents(e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspaseinhibitors); anti-inflammatory agents (e.g., p 38 MAPK inhibitors,TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST,TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidalanti-inflammatory drugs; e.g., TEPDXALIN, TOLMETIN, SUPROFEN);immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors,such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS,EVEROLIMUS); anti-proliferatives (e.g., azathioprine, mycophenolatemofetil); corticosteroids (e.g., prednisolone, hydrocortisone);antibodies such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents (e.g.,heparin, heparin derivatives, urokinase, PPack (dextrophenylalanineproline arginine chloromethylketone), antithrombin compounds, plateletreceptor antagonists, anti-thrombin antibodies, anti-plate let receptorantibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandininhibitors, and platelet inhibitors); and anti-oxidants (e.g., probucol,vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione,L-cysteine, N-acetylcysteine) as well as local anesthetics.

In one example, the cells and/or soluble factors are administered withan immunosuppressive agent or an anti-inflammatory agent or a DMARD or anon-steroidal anti-inflammatory drug.

Exemplary immunosuppressives/anti-inflammatory agents include substancesthat suppress cytokine production, down-regulate or suppressself-antigen expression, or mask the MHC antigens. Examples of suchagents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat.No. 4,665,077) ganciclovir, tacrolimus, glucocorticoids such as cortisolor aldosterone, anti-inflammatory agents such as a cyclooxygenaseinhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptorantagonist; purine antagonists such as azathioprine or mycophenolatemofetil (MMF); alkylating agents such as cyclophosphamide;bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHCantigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypicantibodies for MHC antigens and MHC fragments; cyclosporin A; steroidssuch as corticosteroids or glucocorticosteroids or glucocorticoidanalogs, e.g., prednisone, methylprednisolone, including SOLU-MEDROL®methylprednisolone sodium succinate, and dexamethasone; dihydrofolatereductase inhibitors such as methotrexate (oral or subcutaneous);anti-malarial agents such as chloroquine and hydroxychloroquine;sulfasalazine; leflunomide; cytokine antagonists such as cytokineantibodies or cytokine receptor antibodies includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosisfactor (TNF)-alpha antibodies (infliximab or adalimumab), anti-TNF-alphaimmunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2(IL-2) antibodies and anti-IL-2 receptor antibodies, andanti-interleukin-6 (IL-6) receptor antibodies and antagonists;anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies;anti-L3 T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, such as anti-CD3 or anti-CD4/CD4a antibodies; solublepeptide containing a LFA-3 binding domain (WO 90/08187); streptokinase;transforming growth factor-beta (TGF-beta); streptodomase; RNA or DNAfrom the host; FK506; RS-61443; chlorambucil; deoxyspergualin;rapamycin; T-cell receptor (U.S. Pat. No. 5,114,721); T-cell receptorfragments (W90/11294); BAFF antagonists such as BAFF antibodies and BR3antibodies and zTNF4 antagonists; biologic agents that interfere with Tcell helper signals, such as anti-CD40 receptor or anti-CD40 ligand(CD154), including blocking antibodies to CD40-CD40 ligand and CTLA4-Ig;and T-cell receptor antibodies (EP 340,109) such as T10B9. Someimmunosuppressive agents herein are also DMARDs, such as methotrexate.Examplary immunosuppressive agents herein include cyclophosphamide,chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

Examples of “disease-modifying anti-rheumatic drugs” or “DMARDs” includehydroxycloroquine, sulfasalazine, methotrexate, leflunomide, etanercept,infliximab (plus oral and subcutaneous methotrexate), azathioprine,D-penicillamine, gold salts (oral), gold salts (intramuscular),minocycline, cyclosporine including cyclosporine A and topicalcyclosporine, staphylococcal protein A, including salts and derivativesthereof, etc. In one example, the DMARD is methotrexate

Examples of “non-steroidal anti-inflammatory drugs” or “NSAIDs” includeaspirin, acetylsalicylic acid, ibuprofen, flurbiprofen, naproxen,indomethacin, sulindac, tolmetin, phenylbutazone, diclofenac,ketoprofen, benorylate, mefenamic acid, methotrexate, fenbufen,azapropazone; COX-2 inhibitors such as celecoxib4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonam-ide,valdecoxib, meloxicam, GR 253035 (Glaxo Wellcome); and MK966 (MerckSharp & Dohme), including salts and derivatives thereof, etc.

Alternatively, or additionally, the other compound is an anti-CD20antibody (e.g., rituximab or ofatumumab). Alternatively, oradditionally, the other compound is an anti-CD22 antibody (e.g.,epratuzumab). Alternatively, or additionally, the other compound is ananti-TNF antibody (e.g., infliximab or adalimumab or golimumab) orsoluble TNF receptor (e.g., etanercept). Alternatively, or additionally,the other compound is a CTLA-4 antagonist (e.g., abatacept, CTLA4-Ig).Alternatively, or additionally, the other compound is an anti-IL-6 or ananti-IL-6R antibody (e.g., tocilizumab).

In one example, the STRO-1⁺ cells and/or progeny cells thereof and/orsoluble factors derived therefrom are administered as adjunctive and/orconcomitant therapy to the other therapeutic compound (e.g.,methotrexate).

“Adjunctive therapy” means a treatment that is additional to orsupplements a previous treatment.

“Concomitant therapy” means a treatment that is given at the same timeas another treatment but is not supplemental to the other treatment,e.g., both treatments may individually treat the rheumatic disease.

Discussion herein of co-administering therapeutics or administering morethan one therapeutic does not necessarily mean that the therapeutics areadministered in a single composition. The therapeutics can beadministered simultaneously or sequentially in separate compositions.The period between sequential administration can be several days,provided that there is still sufficient levels of the first therapeuticto provide or add to the therapeutic or prophylactic benefit of thesecond therapeutic when it is administered.

In one example, a pharmaceutical composition as described hereinaccording to any example comprises a compound used to treat inflammatoryjoint disease. Alternatively, a method of treatment/prophylaxis asdescribed herein according to any embodiment additionally comprisesadministering a compound used to treat an inflammatory joint disease(e.g., in the same composition or a separate composition and/or at thesame or different time). Exemplary compounds are described herein andare to be taken to apply mutatis mutandis to these examples of thepresent disclosure.

In another example, a composition as described herein according to anyexample additionally comprises a factor that induces or enhancesdifferentiation of a progenitor cell into a vascular cell. Exemplaryfactors include, vascular endothelial growth factor (VEGF), plateletderived growth factor (PDGF; e.g., PDGF-BB), and FGF.

In another example, a composition as described herein according to anyexample additionally comprises a tissue specific committed cell (TSCC).In this respect, International Patent Application No. PCT/AU2005/001445demonstrates that administration of a TSCC and a STRO-1⁺ cells can leadto enhanced proliferation of the TSCC. In one example, the TSCC is avascular cell. Administration of such a composition to a subject maylead to increased production of vasculature, e.g., leading to increasednutrients being delivered to the affected tissue.

Medical Devices

The present disclosure also provides medical devices for use or whenused in a method as described herein according to any example. Forexample, the present disclosure provides a syringe or catheter or othersuitable delivery device comprising STRO-1⁺ cells and/or progeny cellsthereof and/or soluble factors therefrom and/or a composition asdescribed herein according to any example. Optionally, the syringe orcatheter is packaged with instructions for use in a method as describedherein according to any example.

In another example, the present disclosure provides an implantcomprising STRO-1⁺ cells and/or progeny cells thereof and/or solublefactors therefrom and/or a composition as described herein according toany example. Optionally, the implant is packaged with instructions foruse in a method as described herein according to any example. Suitableimplants may be formed with a scaffold, e.g., as described herein aboveand STRO-1⁺ cells and/or progeny cells thereof and/or soluble factorstherefrom.

Modes of Administration

The STRO-1⁺ cell-derived supernatant or soluble factors, STRO-1⁺ cellsor progeny thereof may be surgically implanted, injected, delivered(e.g., by way of a catheter or syringe), or otherwise administereddirectly or indirectly to the site in need of repair or augmentation,e.g., into a joint or adjacent to the joint.

In one example, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof is/are delivered to the blood stream ofa subject. For example, the STRO-1⁺ cell-derived supernatant or solublefactors, STRO-1⁺ cells or progeny thereof are delivered parenterally.Exemplary routes of parenteral administration include, but are notlimited to, intraperitoneal, intraventricular, intracerebroventricular,intrathecal, intra-arterial, intranodal or intravenous. In one example,the STRO-1⁺ cell-derived supernatant or soluble factors, STRO-1⁺ cellsor progeny thereof are delivered intra-arterially, into an aorta, intoan atrium or ventricle of the heart or into a blood vessel, e.g.,intravenously.

In the case of cell delivery to an atrium or ventricle of the heart,cells can be administered to the left atrium or ventricle to avoidcomplications that may arise from rapid delivery of cells to the lungs.

In one example, the STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are injected into the site of delivery,e.g., using a syringe or through a catheter or a central line.

Selecting an administration regimen for a therapeutic formulationdepends on several factors, including the serum or tissue turnover rateof the entity, the level of symptoms, and the immunogenicity of theentity. For example, an administration regimen maximizes the amount oftherapeutic compound delivered to the patient consistent with anacceptable level of side effects. Accordingly, the amount of formulationdelivered depends in part on the particular entity and the severity ofthe condition being treated.

In one example, STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are delivered as a single bolus dose.Alternatively, STRO-1⁺ cell-derived supernatant or soluble factors,STRO-1⁺ cells or progeny thereof are administered by continuousinfusion, or by doses at intervals of, e.g., one day, one week, or 1-7times per week. An exemplary dose protocol is one involving the maximaldose or dose frequency that avoids significant undesirable side effects.A total weekly dose depends on the type and activity of the compoundbeing used. Determination of the appropriate dose is made by aclinician, e.g., using parameters or factors known or suspected in theart to affect treatment or predicted to affect treatment. Generally, thedose begins with an amount somewhat less than the optimum dose and isincreased by small increments thereafter until the desired or optimumeffect is achieved relative to any negative side effects. Importantdiagnostic measures include those of symptoms of diabetes.

The present inventors have shown therapeutic benefits provided bySTRO-1⁺ cells and/or progeny thereof and/or soluble factors derivedtherefrom are observed for at least four weeks in a subject.Accordingly, in some examples the cells are administered weekly,fortnightly, once every three weeks or once every four weeks.

In accordance with examples of the invention directed to treating ordelaying the progression of an inflammatory joint disease, in oneexample, the STRO-1⁺ cells and/or progeny cells thereof and/or solublefactors derived therefrom are administered following diagnosis of thedisorder, e.g., using standard methods known in the art and/or describedherein.

For those examples directed to preventing or delaying the onset of aninflammatory joint disease, the STRO-1⁺ cells and/or progeny cellsthereof and/or soluble factors derived therefrom can be administeredprior to clinical diagnosis of the disorder.

Patient Populations

The methods of present disclosure are also useful for treating subjectsfalling within sub-populations of subjects suffering from a rheumaticdisease.

In one example, the subject suffers from rheumatic disease, e.g.,rheumatoid arthritis, and does not respond adequately to a TNF inhibitor(e.g., an anti-TNF antibody or a soluble TNF receptor). A subject who“does not respond adequately to a TNF inhibitor” has experienced aninadequate response to previous or current treatment with one or moreTNF inhibitors because of toxicity or inadequate efficacy. In oneexample, such patient has received, for example, etanercept for >3months at 25 mg twice a week or at least 4 infusions of infliximab at >3mg/kg but has had an inadequate response thereto.

In one example, the subject suffers from rheumatic disease, e.g.,rheumatoid arthritis, and does not respond adequately to methotrexate. Asubject who “does not respond adequately to methotrexate” is a patientwho has experienced an inadequate response to previous or currenttreatment with methotrexate because of toxicity or inadequate efficacy.In one example, the patient has been on methotrexate (10-25 mg/week) forat least 12 weeks and still has active disease.

In one example, the subject is already receiving treatment withmethotrexate.

In one example, the subject is already receiving treatment withmethotrexate and administration of the STRO-1⁺ cells and/or progenycells thereof and/or soluble factors derived therefrom delaysprescription of an anti-TNF therapy compared to a subject who has notreceived the STRO-1⁺ cells and/or progeny cells thereof and/or solublefactors derived therefrom.

In one example, the subject suffers from active rheumatoid arthritis. Asubject with “active rheumatoid arthritis” means a subject with activeand not latent symptoms of rheumatoid arthritis. In one example, suchpatient has moderate-to-severe active rheumatoid arthritis of >6 monthsdisease duration at time of baseline visit. In one example, suchpatients will have: (1) swollen joint count (SJC)>4 (66 joint count),(2) tender joint count (TJC)>4 (68 joint count), and/or C-reactiveprotein (CRP)>upper limit of normal (ULN) at screening visit.

In one example, the subject suffers from moderate active rheumatoidarthritis or severe active rheumatoid arthritis or moderate to severeactive rheumatoid arthritis. A person having moderate active rheumatoidarthritis generally has a combination of at least two or three or fouror all of the following symptoms:

-   -   Between 6 and 20 inflamed joints    -   Usually no inflammation in tissues other than the joints    -   An elevated ESR or CRP levels    -   A positive rheumatoid factor test or anti-cyclic citrullinated        peptide (anti-CCP) antibodies    -   Evidence of inflammation but no evidence of bone damage on        x-rays

A person having severe active rheumatoid arthritis generally has acombination of at least two or three or four or all of the followingsymptoms:

-   -   More than 20 persistently inflamed joints or a rapid loss of        functional abilities    -   Elevated ESR or CRP levels    -   Anemia related to chronic illness    -   Low blood albumin level    -   A positive rheumatoid factor test, often with a high level    -   Evidence of bone and cartilage damage on x-ray    -   Inflammation in tissues other than joints

In one example, a subject has persistently active rheumatoid arthritis.A person with persistently active rheumatoid arthritis has had evidenceof inflammation for at least six to twelve months and may haveirreversible joint damage and loss of function.

In one example, administration of the cells or soluble factors inhibitsprogression of structural joint damage. The expression “inhibitingprogression of structural joint damage” in a subject refers topreventing or slowing structural joint damage caused by a rheumaticdisease, e.g., a subject suffering from rheumatoid arthritis for examplebased on eroded joint count and/or joint damage score. Methods formeasuring progression of structural joint damage are known to theskilled person, and include, without limitation Genant-modified TotalSharp Score (TSS), erosion score (ES), and/or joint space narrowing(JSN) score. In one example, a method disclosed herein additionallycomprises assessing progression of structural joint damage, e.g., usinga method described herein and/or by X-ray. For example, the assessmentis made about 1 month or 3 months or 6 months or 12 months after thelast administration of the cells or soluble factors.

The present invention is described further in the following non-limitingexamples.

EXAMPLES Example 1: Immunoselection of MPCs by Selection of STRO-3⁺Cells

Bone marrow (BM) is harvested from healthy normal adult volunteers(20-35 years old). Briefly, 40 ml of BM is aspirated from the posterioriliac crest into lithium-heparin anticoagulant-containing tubes.

BMMNC are prepared by density gradient separation using Lymphoprep™(Nycomed Pharma, Oslo, Norway) as previously described (Zannettino, A.C. et aL (1998) Blood 92: 2613-2628). Following centrifugation at 400×gfor 30 minutes at 4° C., the buffy layer is removed with a transferpipette and washed three times in “HHF”, composed of Hank's balancedsalt solution (HBSS; Life Technologies, Gaithersburg, Md.), containing5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

STRO-3⁺ (or TNAP⁺) cells were subsequently isolated by magneticactivated cell sorting as previously described (Gronthos et al. (2003)Journal of Cell Science 116: 1827-1835; Gronthos, S. and Simmons, P. J.(1995) Blood 85: 929-940). Briefly, approximately 1-3×10⁸ BMMNC areincubated in blocking buffer, consisting of 10% (v/v) normal rabbitserum in HHF for 20 minutes on ice. The cells are incubated with 200 μlof a 10 μg/ml solution of STRO-3 mAb in blocking buffer for 1 hour onice. The cells are subsequently washed twice in HHF by centrifugation at400×g. A 1/50 dilution of goat anti-mouse γ-biotin (SouthernBiotechnology Associates, Birmingham, UK) in HHF buffer is added and thecells incubated for 1 hour on ice. Cells are washed twice in MACS buffer(Ca²⁺- and Mn²⁺-free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01%sodium azide) as above and resuspended in a final volume of 0.9 ml MACSbuffer.

One hundred μl streptavidin microbeads (Miltenyi Biotec; BergischGladbach, Germany) are added to the cell suspension and incubated on icefor 15 minutes. The cell suspension is washed twice and resuspended in0.5 ml of MACS buffer and subsequently loaded onto a mini MACS column(MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml MACSbuffer to retrieve the cells which did not bind the STRO-3 mAb(deposited on 19 Dec. 2005 with American Type Culture Collection (ATCC)under accession number PTA-7282—see International Publication No. WO2006/108229). After addition of a further 1 ml MACS buffer, the columnis removed from the magnet and the TNAP⁺ cells are isolated by positivepressure. An aliquot of cells from each fraction can be stained withstreptavidin-FITC and the purity assessed by flow cytometry.

Example 2: Cells Selected by STRO-3 mAb are STRO-1^(bright) Cells

Experiments were designed to confirm the potential of using STRO-3 mAbas a single reagent for isolating cells STRO-1^(bright) cells.

Given that STRO-3 (IgG1) is a different isotype to that of STRO-1 (IgM),the ability of STRO-3 to identify clonogenic CFU-F was assessed bytwo-color FACS analysis based on its co-expression with STRO-1⁺ cellsisolated using the MACS procedure (FIG. 1). The dot plot histogramrepresents 5×10⁴ events collected as listmode data. The vertical andhorizontal lines were set to the reactivity levels of <1.0% meanfluorescence obtained with the isotype-matched control antibodies, 1B5(IgG) and 1A6.12 (IgM) treated under the same conditions. The resultsdemonstrate that a minor population of STRO-1^(bright) cellsco-expressed TNAP (upper right quadrant) while the remaining STRO-1⁺cells failed to react with the STRO-3 mAb. Cells isolated by FACS fromall four quadrants were subsequently assayed for the incidence of CFU-F(Table 1).

TABLE 1 Enrichment of human bone marrow cells by dual-colour FACSanalysis based on the co-expression of the cell surface markers STRO-1and TNAP (refer to FIG. 1). FACS sorted cells were cultured understandard clonogenic conditions in alpha MEM supplemented with 20% FCS.The data represents the mean number of day 14 colony-forming cells(CFU-F) per 10⁵ cells plated ± SE (n = 3 different bone marrowaspirates). These data suggest that human MPC are exclusively restrictedto the TNAP positive fraction of BM which co-express the STRO-1 antigenbrightly. Frequency of Enrichment Bone Marrow Fraction CFU-F/10⁵ Cells(Fold Increase) Unfractionated BMMNC  11.0 ± 2.2 1.0TNAP⁺/STRO-1^(bright) 4,511 ± 185 410 TNAP⁺/STRO-1^(dull) 0.0 0.0

Example 3: Relative Gene and Surface Protein Expression of STRO-1^(dull)and STRO-1^(bright) Cells

In the first series of experiments, semi-quantitative RT-PCR analysiswas employed to examine the gene expression profile of variouslineage-associated genes expressed by STRO-1^(dull) or STRO-1^(bright)populations, isolated by fluorescence activated cell sorting (FIG. 2A).In the second series of experiments, flow cytometry and mean channelfluorescence analysis was employed to examine the surface proteinexpression profile of various lineage-associated proteins expressed bySTRO-1^(dull) or STRO-1^(bright) populations, isolated by fluorescenceactivated cell sorting.

Total cellular RNA was prepared from either 2×10⁶ STRO-1^(bright) orSTRO-1^(dull) sorted primary cells, chondrocyte pellets and otherinduced cultures and lysed using RNAzolB extraction method (Biotecx Lab.Inc., Houston, Tex.), according to the manufacturer's recommendations.RNA isolated from each subpopulation was then used as a template forcDNA synthesis, prepared using a First-strand cDNA synthesis kit(Pharmacia Biotech, Uppsala, Sweden). The expression of varioustranscripts was assessed by PCR amplification, using a standard protocolas described previously (Gronthos et al., J. Bone and Min. Res.14:48-57, 1999). Primer sets used in this study are shown in Table 2.Following amplification, each reaction mixture was analyzed by 1.5%agarose gel electrophoresis, and visualized by ethidium bromidestaining. RNA integrity was assessed by the expression of GAPDH.

Relative gene expression for each cell marker was assessed withreference to the expression of the house-keeping gene, GAPDH, usingImageQant software (FIG. 2B, C). In addition, dual-colour flowcytometric analysis was used to examine the protein expression profileof ex vivo expanded MPC based on their expression of a wider range ofcell lineage-associated markers in combination with the STRO-1 antibody.A summary of the general phenotype based on the gene and proteinexpression of STRO-1^(dull) and STRO-1^(bri) cultured cells is presentedin Table 3. The data indicate that ex vivo expanded STRO-1^(bright) MPCexhibit differentially higher expression of markers associated withperivascular cells, including angiopoietin-1, VCAM-1, SDF-1, TNFα, andRANKL. Comparisons between the protein and gene expression profiles ofSTRO-1^(dull) and STRO-1^(bright) cultured cells are summarized inTables 3 and 4.

Subtractive hybridization studies were also performed in order toidentify genes uniquely expressed by STRO-1^(bri) cells. Briefly,STRO-1^(dull) and STRO-1^(bright) were isolated as described above (seeFIG. 3A). Total RNA was prepared from STRO-1^(dull) and STRO-1^(bright)cells pooled from 5 different marrow samples using the RNA STAT-60system (TEL-TEST). First-strand synthesize was performed using the SMARTcDNA synthesis kit (Clontech Laboratories). The resultantmRNA/single-stranded cDNA hybrid was amplified by long-distance PCR(Advantage 2 PCR kit; Clontech) using specific primer sites at the 3′and 5′ prime ends formed during the initial RT process according to themanufacturer's specifications. Following RsaI digestion of theSTRO-1^(bright) cDNA, 2 aliquots were used to ligate different specificadaptor oligonucleotides using the Clontech PCR-Select cDNA SubtractionKit. Two rounds of subtractive hybridization were performed usingSTRO-1^(bright) (tester) and STRO-1^(dull) (driver) cDNA, and viceversa, according to the manufacturer's protocol. This procedure was alsoperformed in reverse using STRO-1^(dull) tester cDNA hybridized againstSTRO-1^(bright) driver cDNA.

To identify genes uniquely expressed by STRO-1^(bright) population,STRO-1^(bright)-subtracted cDNA was used to construct replicatelow-density microarray filters comprising 200 randomly selectedbacterial clones transformed with the STRO-1^(bright) subtracted cDNAsligated into a T/A cloning vector. The microarrays were subsequentlyprobed with either [³²P] dCTP-labeled STRO-1^(bright) or STRO-1^(dull)subtracted cDNA (FIG. 3B-C). Differential screening identified a totalof 44 clones, which were highly differentially expressed between theSTRO-1^(dull) and STRO-1^(bright) subpopulations. DNA sequencing of allthe differentially expressed clones revealed that only 1 clone wasrepresentative of a known stromal cell mitogen; namely, platelet-derivedgrowth factor (PDGF) (Gronthos and Simmons, Blood. 85: 929-940, 1995).Interestingly, 6 of the 44 clones were found to contain DNA insertscorresponding to the chemokine, stromal-derived factor-1 (SDF-1). Thehigh abundance of SDF-1 transcripts in human STRO-1^(bright) cells wasconfirmed by semiquantitative RT-PCR of total RNA prepared from freshlysorted STRO-1^(bright), STRO-1^(dull), and STRO-1^(negative) bone marrowsubpopulations (FIG. 3D and Table 3).

TABLE 2  RT-PCR primers and conditions for the specificamplification of human mRNA Target Product GeneSense/Antisense (5′-3′) Primer Sequences Size GAPDHCACTGACACGTTGGCAGTGG (SEQ ID NO: 1) 417CATGGAGAAGGCTGGGGCTC (SEQ ID NO: 2) SDF-1GAGACCCGCGCTCGTCCGCC (SEQ ID NO: 3) 364GCTGGACTCCTACTGTAAGGG (SEQ ID NO: 4) IL-1βAGGAAGATGCTGGTTCCCTCTC (SEQ ID NO: 5) 151CAGTTCAGTGATCGTACAGGTGC (SEQ ID NO: 6) LT-1TCACTATGGAAGATCTGATTTCTTACAGT (SEQ ID NO: 7) 380GGTATAAATACACATGTGCTTCTAG (SEQ ID NO: 8) TNF-αTCAGATCATCTTCTCGAACC (SEQ ID NO: 9) 361CAGATAGATGGGCTCATACC (SEQ ID NO: 10) KDRTATAGATGGTGTAACCCGGA (SEQ ID NO: 11) 450TTTGTCACTGAGACAGCTTGG (SEQ ID NO: 12) RANKLAACAGGCCTTTCAAGGAGCTG (SEQ ID NO: 13) 538TAAGGAGGGGTTGGAGACCTCG (SEQ ID NO: 14) LeptinATGCATTGGGAACCCTGTGC (SEQ ID NO: 15) 492GCACCCAGGGCTGAGGTCCA (SEQ ID NO: 16) CBFA-1GTGGACGAGGCAAGAGTTTCA (SEQ ID NO: 17) 632TGGCAGGTAGGTGTGGTAGTG (SEQ ID NO: 18) PPARγ2AACTGCGGGGAAACTTGGGAGATTCTCC (SEQ ID NO: 18) 341AATAATAAGGTGGAGATGCAGGCTCC (SEQ ID NO: 19) OCNATGAGAGCCCTCACACTCCTC (SEQ ID NO: 20) 289CGTAGAAGCGCCGATAGGC (SEQ ID NO: 21) MyoDAAGCGCCATCTCTTGAGGTA (SEQ ID NO: 22) 270GCGAGAAACGTGAACCTAGC (SEQ ID NO: 23) SMMHCCTGGGCAACGTAGTAAAACC (SEQ ID NO: 24) 150TATAGCTCATTGCAGCCTCG (SEQ ID NO: 25) GFAPCTGTTGCCAGAGATGGAGGTT (SEQ ID NO: 26) 370TCATCGCTCAGGAGGTCCTT (SEQ ID NO: 27) NestinGGCAGCGTTGGAACAGAGGTTGGA (SEQ ID NO: 28) 460CTCTAAACTGGAGTGGTCAGGGCT (SEQ ID NO: 29) SOX9CTCTGCCTGTTTGGACTTTGT (SEQ ID NO: 30) 598CCTTTGCTTGCCTTTTACCTC (SEQ ID NO: 31) CollagenAGCCAGGGTTGCCAGGACCA (SEQ ID NO: 32) 387 type XTTTTCCCACTCCAGGAGGGC (SEQ ID NO: 33) AggrecanCACTGTTACCGCCACTTCCC (SEQ ID NO: 34) 184ACCAGCGGAAGTCCCCTTCG (SEQ ID NO: 35)

TABLE 3 Summary of the Relative Gene Expression in STRO-1^(Bright) andSTRO-1^(Dull) populations. A list of genes which displayed measurableand differential expression between the STRO-1^(Bright) andSTRO-1^(Dull) populations as determined by reverse transcription-PCR arepresented. Values represent the relative gene expression with referenceto the house-keeping gene, GAPDH. Gene Expression relative to GAPDHSTRO- STRO- Tissue Marker 1^(Bright) 1^(Dull) Neurons GFAP (GlialFibrillary Acidic 0.1 0.7 Protein) Bone OCN (Osteocalcin) 1.1 2.5 OSX(Osterix) 0.4 1.3 CBFA-1 (Core Factor Binding 0.3 0.6 Protein-1)Immunoregulatory RANKL (Receptor Activator 1.6 0.3 of Nuclear Factor κB) SDF-1-alpha (Stromal 3.2 0.1 Derived factor-1-alpha) Fat Leptin 3.14.2 Cardiomyocytes GATA-4 1.1 2.9 Enthothelial cells Ang-1(Angiopoietin-1) 1.5 0.8 Chondrocytes Sox 9 0.3 1.1 COL X (Collagen X)3.5 2.8 Pro-inflammatory TNF-alpha (Tumour necrosis 1.7 0.9 Cytokinesalpha)

To correlate protein surface expression with density of STRO-1expression, single cell suspensions of ex vivo expanded cells derivedbone marrow MPC were prepared by trypsin/EDTA detachment andsubsequently incubated with the STRO-1 antibody in combination withantibodies identifying a wide range of cell lineage-associated markers.STRO-1 was identified using a goat anti-murine IgM-fluoresceinisothiocyanate while all other markers were identified using either agoat anti-mouse or anti-rabbit IgG-phycoerythrin. For those antibodiesidentifying intracellular antigens, cell preparations were first labeledwith the STRO-1 antibody, fixed with cold 70% ethanol to permeabilizethe cellular membrane and then incubated with intracellularantigen-specific antibodies. Isotype matched control antibodies wereused under identical conditions. Dual-colour flow cytometric analysiswas performed using a COULTER EPICS flow cytometer and list mode datacollected. The dot plots represent 5,000 listmode events indicating thelevel of fluorescence intensity for each lineage cell marker (y-axis)and STRO-1 (x-axis). The vertical and horizontal quadrants wereestablished with reference to the isotype matched negative controlantibodies.

TABLE 4 Summary of the Relative Protein Expression in STRO-1^(Bright)and STRO-1^(Dull) populations. A list of proteins which displayeddifferential expression between the STRO-1^(Bri) and STRO-1^(Dull)populations as determined by flow cytometry are presented. Valuesrepresent the relative mean fluorescence intensity of staining. MeanFluorescence Intensity STRO- STRO- Tissue Marker 1^(Bright) 1^(Dull)Neurons Neurofilament 1.7 20.5 Bone ALK PHOS (Alkaline 5.7 44.5Phophatase) Immunoregulatory RANKL (Receptor Activator 658.5 31.0 ofNuclear Factor κ B) Epithelial Cells CytoKeratin 10 + 13 1.2 23.3Cytokeratin 14 1.8 8.8 Smooth Muscle α-SMA (Alpha Smooth 318.0 286.0Muscle Actin) Chondrocytes Byglycan 84.4 65.9 Basal Fibroblast TenascinC 22.2 6.9 Cardiomyocyte Troponin C 2.5 15.0

Example 4: A Sheep Model of Rheumatoid Arthritis

4.1 Methods

An outline of the method for producing a sheep model of rheumatoidarthritis is as follows:

-   -   1. Day 0        -   i. 5 sheep (B1626, B1584, B3619, B1612, B1302) were            administered Freund's complete adjuvant+5 mg Bovine type II            collagen (5×0.2 ml S/C per sheep). Solution was administered            subcutaneously (S/C)        -   ii. 2 sheep (B1627, B4036) were administered Freund's            complete adjuvant+5 mg Chicken type II collagen (5×0.2 ml            S/C per sheep)        -   iii. 10 ml of blood was collected for testing for Collagen            type II antibodies using an ELISA        -   iv. Clinical examination for the following:            -   Lameness            -   Swelling            -   Joint thickening    -   2. Day 14        -   i. 5 sheep were administered Freund's incomplete adjuvant+5            mg. Bovine type II collagen (5×0.2 ml S/C per sheep)        -   ii. 2 sheep were administered Freund's incomplete adjuvant+5            mg Chicken II collagen (5×0.2 ml S/C per sheep)        -   iii. 10 ml of blood was collected for testing for Collagen            type II antibodies using an ELISA and white blood cell            counts        -   iv. Clinical examination for the following:            -   Lameness            -   Swelling            -   Joint thickening    -   3. Day 28        -   i. 5 sheep were administered 100 μg bovine type II collagen            in saline via intra-articular injection into left hock (500            μl per sheep)        -   ii. 2 sheep 100 μg chicken type H collagen intra-articular            injection in saline into left hock (500₁11 per sheep)        -   iii. 10 ml of blood was collected for testing for Collagen            type II antibodies using an ELISA and white blood cell            counts        -   iv. Clinical examination for the following:            -   Lameness            -   Swelling            -   Joint thickening    -   4. Day 30+2 days IA injection        -   i. Clinical examination for the following:            -   Lameness            -   Swelling            -   Joint thickening    -   5. Day 36+8 days IA injection        -   i. Clinical examination for the following:            -   Lameness            -   Swelling            -   Joint thickening    -   6. Day 42+14 days IA injection        -   i. 4 sheep (B1626, B3619, B1612, B1302) receiving the bovine            type II collagen were killed        -   ii. 2 sheep (B1627, B4036) receiving the chicken type II            collagen were killed        -   iii. 10 ml of blood was collected for testing for Collagen            type II antibodies using an ELISA and white blood cell            counts        -   iv. Clinical examination for the following:            -   Lameness            -   Swelling            -   Joint thickening        -   v. Killed sheep            -   Synovial fluid left & right hock joints                -   a. Assessed for collagen type II antibodies                -   b. Cell counts            -   Synovial tissue left & right hock joints                -   a. Formalin fixation. H&E sections                -   b. Liquid nitrogen freezing            -   Articular Cartilage left and right                -   a. Photos                -   b. Formalin fixation and de-calcification for H & E    -   7. Day 56        -   i. 1 sheep (1584) receiving bovine collagen type II killed        -   ii. 10 ml of blood was collected for testing for Collagen            type II antibodies using an ELISA and white blood cell            counts        -   iii. Clinical examination for the following            -   Lameness            -   Swelling            -   Joint thickening        -   iv. Killed sheep            -   Synovial fluid left & right hock joints                -   a. Assessed for collagen type II antibodies Cell                    counts            -   Synovial tissue left & right hock joints                -   a. Formalin fixation H&E sections                -   b. Liquid nitrogen freezing            -   Articular Cartilage L & R                -   a. Photos                -   b. Formalin fixation and de-calcification for H & E

4.2 Results

Clinical signs of mild lameness were evident in all 7 sheep, with jointswelling and pain on joint flexion apparent in 4 sheep. These signs wereobserved in the left (treated) hock only.

After the sheep were euthanased, gross thickening of the synoviumsurrounding the tibiotarsal joint was evident in 6 of the 7 sheep. Thiswas the most striking evidence of inflammatory changes. Examining thearticular cartilage of this joint, gross inflammatory erosive lesionswere observed in 3 of the sheep, most notably on the articular surfaceof the talus bone.

Where inflammatory changes were observed in the left (treated) hock, inthe contralateral (right) hock very mild inflammatory changes wereoccasionally observed.

Table 5 summarizes results of features observed in treated sheep.

TABLE 5 Summary of the clinical, gross pathological andhistopathological features observed in the 7 sheep Sheep B1626 B1584B1627 B4036 B3619 B1612 B1302 Treatment BCII BCII CCII CCII BCII BCIIBCII Length of treatment 42 d 56 d 42 d 42 d 42 d 42 d 42 d ClinicalAssesment Lameness ++ + ++ + ++ ++ + Joint swelling + 0 ++ 0 ++ ++ 0Pain on flexion + 0 + 0 + + 0 Necropsy Synovium + + ++ + ++ ++ −Thickening Thickening Thickening Thickening Thickening Thickening NormalArticular Cartilage No gross No gross Cartilage No gross CartilageCartilage No gross lesions lesions lesion lesions lesion lesion lesionsHistopathology 5 3 6 6 9 9 4 Synovitis grading/9 (see Table 7) SynoviumSynovial membrane Early signs of Marked stromal Fibrin/collagen?Synovial Synovial Mild moderately inflammation, thickening anddeposition in membrane membrane synovial thickened. Marked includingfibrosis, synovium, many thickening and markedly thickening inflammatorycell perivascular increased fibroblasts. fibrosis. Stromal thickened byand fibrosis. infiltrate, lymphocytes. vascularity Perivascular fibrosisand fibrosis. lymphocytes and Synovial Synovial leukocyte perivascularMarked cellular plasma cells. membrane is membrane aggregates, withlymphocyte infiltrate slightly thickened. lymphocytes and accumulationin stroma and thickened lymphocytic plasma cells. some areas ofperivascular necrosis. infiltrate. Articular Cartilage No cartilage Nocartilage Full thickness No cartilage Full thickness Full thickness Nocartilage lesions evident lesions evident erosion and lesions evidenterosions and also inflammatory lesions evident on section on sectioninflammation on section partial thickness erosion, on section inflamedareas lymphocytes and macrophages

Clinical Assessments of Sheep

Table 6 sets out the scoring system used to assess treated sheep.

TABLE 6 Clinical scoring Lameness Score Behaviour Standing posture Gait0 Behaviour unaffected Sheep stands squarely on all 4 legs Even strides;able to change direction rapidly 1 Sheep stands squarely on all 4 legsAbnormal stride length (not easily identified). Movement no longerfluent. Still able to change direction easily. 2 Uneven postureShortened stride. Lameness detected in a straight line. Awkward changingdirection 3 Uneven posture. Shortened stride. Minimum weight Sheep willnot bear weight on bearing on affected limb affected limb. 4 Will try toremain separate Affected limb elevated off floor Sheep may not placeaffected limb to the from others in the group ground when moving 5 Willnot stand unaided Does not move. Score Joint swelling Joint pain onflexion 0 None detectable None elicited 1 Barely detectable but presentSlight discomfort on strong flexion 2 Clearly discernible swelling onpalpation Clear discomfort with strong flexion 3 Very marked jointswelling Severe discomfort even with slight flexion; sheep veryreluctant to flex joint

Tables 7-11 show results of clinical assessment of sheep at varioustimepoints. No clinical changes observed until after IA injection ofCII.

TABLE 7 Clinical assessment (+2 d IA) 1626 1584 1627 4036 3619 1612 1302BII BII CII CII BII BII BII Lameness 1/5 2/5 3/5 3/5 2/5 3/5 2/5 Joint0/3 0/3 1/3 1/3 1/3 2/3 0/3 swelling Joint 0/3 0/3 2/3 2/3 2/3 2/3 2/3pain on flexion Other — Stiff Heat heat Less Heat -heat slight Heat

TABLE 8 Clinical assessment (+8 d IA) 1626 1584 1627 4036 3619 1612 1302BII BII CII CII BII BII BII Lameness 1/5 1/5 2/5 1/5 0/5 1/5 0/5 Joint1/3 0/3 1/3 1/3 0/3 1/3 1/3 swelling Joint 1/3 1/3 2/3 1/3 0/3 1/3 0/3pain on flexion Other Slight Heat Heat Slight — Heat Slight heat heatheat

TABLE 9 Clinical assessment (+14 d IA) 1626 1584 1627 4036 3619 16121302 BII BII CII CII BII BII BII Lameness 2/5 1/5 2/5 1/5 2/5 2/5 1/5Joint 1/3 0/3 2/3 0/3 2/3 2/3 0/3 swelling Joint 1/3 0/3 1/3 0/3 1/3 1/3 0/30 pain on flexion Necropsy Slightly Ongoing Synovial slight SynovialSynovial Normal Results thickened thickening thickening thickeningthickening gross synovium + ++ + ++ ++ appearance Articular No grossCartilage No gross Lesion Cartilage No gross cartilage lesions erosionslesions (erosion) lesions lesions

TABLE 10 Clinical assessment (+21 d IA) 1584 21 d IA BII Lameness 0/5Joint 1/3 swelling Joint pain 0/3 on flexion Other

TABLE 11 (+28 d IA) 1584 28 d IA BII Lameness 1/5 Joint 1/3 swellingJoint pain 0/3 on flexion Necropsy thickening Results + synoviumArticular No gross cartilage lesions

FIG. 4 shows the mean lameness score for sheep following intrarticularcollagen type II injection. As shown lameness increased at about 28 daysafter the first collagen type II injection.

FIG. 5 shows leukocyte counts in synovial fluid from right(unstimulated) and let (stimulated) hocks of individual sheep. In thehigher responders the leukocyte concentration in the control side wasalso raised indicating the possibility of a systemic response. FIG. 6shows mean leukocyte counts in the synovial fluid of control sheep andsheep immunized with bovine type II collagen.

FIGS. 7A and 7B show levels of IgM and IgG antibodies in synovial fluidfrom sheep immunized with bovine type II collagen and chicken type IIcollagen, respectively. These results indicate increased IgM and IgGagainst type II collagen in immunized hocks of these animals. These dataare also summarized in Table 12.

TABLE 12 Relative levels of antibodies to bovine or chicken Type IIcollagen in the plasma of immunised sheep Synovitis score* Left hock/Plasma IgM: day 14 Plasma IgG: day 28 Sheep Right hock Endpoint titre**Endpoint titre Bovine B1626 5/4 9 × 10³ 9 × 10⁴ B1302 4/3 9 × 10³ 9 ×10⁴ B1584  3/2.5 1 × 10⁴ 9 × 10⁴ B1612 9/5 1 × 10⁴ 9 × 10⁶ B3619 9/4 1 ×10⁴ 9 × 10⁶ Chicken B1627 6/3 9 × 10⁴ 1 × 10⁷ B4036 6/3 9 × 10⁴ 1 × 10⁷*The grading system incorporates three morphological criteria:hyperplasia of the synovial cell layer, inflammatory infiltration andactivation of the synovial stroma and has a maximum score of 9 (Krenn etal., Pathol Res. Pract. 198: 317-325, 2002). **IgM levels were highestat day 14.

The data presented above show that collagen induced arthritis (CIA) wasinduced in 7 sheep by the subcutaneous injection of heterologous type IIcollagen in Freund's adjuvant on day 0.0 and day 14 and theintra-articular injection of type II collagen into the hock joint on day28. The disease progressed rapidly and by 14 days after theintra-articular injection of CII was characterized by jointinflammation, synovial hyperplasia, mononuclear cell infiltration, anticollagen type II antibodies and in some sheep erosive lesions of thearticular cartilage. Clinical signs of lameness were evident in all 7sheep and gross thickening of the synovium surrounding the tibiotarsaljoint was evident in 6 of the 7 sheep.

CIA in sheep appears to be an excellent large animal model of arthritiswith significant similarities to human rheumatoid arthritis.

Example 5: Experimental Design

A sheep model of rheumatoid arthritis is produced essentially asdescribed in Example 4.

Thirty-six sheep will be randomly allocated to one of six (6) groups,each including eight (6) sheep, as shown in Table 13. Test articles willbe administered to relevant animals 42 days post the first collageninjection.

Thirty days after the administration of the test articles, the sheepwill be euthanised and gross necropsy examination conducted and tissuecollected for pathology and histological examination.

TABLE 13 Treatment Assignment Treatment Group No. of MPC ID animals TypeDose Route Timing Sacrifice A 6 oMPCs 0.3 IV Day 42 Day 72 Million/kg B6 oMPCs 1.0 IV Day 42 Day 72 Million/kg C 6 oMPCs 2.0 IV Day 42 Day 72Million/kg D 6 Control N/A IV Day 42 Day 72 (saline) E 6 oMPCs 25 Intra-Day 42 Day 72 Million articular F 6 Control N/A Intra- Day 42 Day 72(saline) articular

Preparation of MPC Doses

-   -   24 million ovine MPCs (oMPCs) in 4.0 mL        ProFreeze®/DMSO/Alpha-MEM (to be used to provide a dose of 0.3        million oMPCs/kg by IV injection)    -   80 million oMPCs in 4.0 mL ProFreeze®/DMSO/Alpha-MEM (to be used        to provide a dose of 1 million oMPCs/kg by IV injection)    -   160 million oMPCs in 4.0 mL ProFreeze®/DMSO/Alpha-MEM (to be        used to provide a dose of 2 million oMPCs/kg by IV injection)    -   35 million oMPCs in 0.7 mL ProFreeze®/DMSO/Alpha-MEM (to be used        to provide a dose of 25 million oMPCs by intra-articular        injection)    -   Sterile saline (control).

Gross Pathology

Necropsy and tissue collection is performed on all animals that die orare euthanised. Collected tissues is fixed in 10% buffered formalin.Macroscopic findings are recorded for each tissue.

The left (treated) and right (control) hock joints are exposed anddissected down to the synovial membrane. Samples of synovium (andunderlying fat) is placed in either 10% buffered neutral formalin forfixing or OCT compound for snap freezing.

The joints are then disarticulated and the articular surfaces examinedfor evidence of gross cartilage lesions and photographed. Segments ofthe articular surface of the talus bone (see below) are removed using asaw, and fixed in 10% buffered neutral formalin.

Histopathological Scoring of Synovial Tissues from Hock Joints

The methodology for the preparation of histological sections and scoringof pathological changes within the synovial membrane and subsynovialtissues of the left and right hock joint of control and treated animalswas based on the publication of Krenn et al (Pathol Res. Pract.198:317-325).

Immunohistochemical Studies and Scoring of Synovial Tissues from HockJoints

Synovial membrane was collected from the dorso-lateral and dorso-medialregion of the right and left tibiotarsal (Hock) joints from treated andcontrol animals. These tissues were frozen in OCT compound and sectionedusing a cryostat onto glass slides. Sections were subjected to stainingusing standard immunohistochemical methods for the identification ofspecific cell types and antibodies following published methodologies(16) and commercially available protocols. The antibodies used includedthose raised against CD4 (44-97), CD8 (38-65), gamma-delta (γδ T cells(86D/127-5), CD14 (VMRD a-M-M9), B cell (CD79a: HM57) and the cytokinesTNF-alpha, Interleukin-6, interleukin-1 beta, interleukin-17,Interleukin-10, Interferon gamma, Factor VIII and VCAM-1 and wereobtained from commercial sources or prepared in house. The stainedsections were scored blinded.

Clinical Pathology

Approximately 30-40 mL of blood was collected at pre-study entry, Day 0(Baseline), Day 42, Day 49, Day 56, Day 63 and at necropsy (Day 72) andused for the following assays:

Cytokines:

The levels of the cytokines, TNF-α, IFN-g, IL-1b and IL-6, in synovialfluid and plasma were determined.

Statistical Methods

Statistical analysis and graphical representation of the data obtainedfor the various treatment groups was undertaken using one way ANOVAfollowed by Tukey's or Newman-Keuls multiple comparison tests usingGraphpad Prism Statistical software (version 5.0b) (GraphPad SoftwareInc, La Jolla, Calif. USA). Statistical significance between treatmentgroups was taken as p<0.05. For parametric data significance betweenindividual treatment groups and controls was determined using the pairedStudent's t-test. For non-parametric assessment, differences betweengroups were evaluated using the Mann-Whitney or the Wilcoxonmatched-pairs signed rank tests with significance accepted at p<0.05.

Example 6: Results of Histopathological Scoring of Synovial Tissues fromHock Joints

The individual histopathological scores for the synovial membranes ofleft (bovine type II collagen (BII) injected joint) and right flockjoints of all animals were determined by blinded observers. Theaggregate and component score for each section obtained by summation ofthe individual scores for hyperplasia, stromal (synovial tissue)activation, and inflammatory infiltrate are summarised graphically inFIGS. 8A and 8B. From these figures it is evident that the aggregatescore for the BII injected left joints was higher than for the scoresfor the contralateral right joints. Moreover, statistically significantdifferences between the MPC treated groups and the control group weredemonstrated for the low (p<0.017) and high (p<0.025) MPC injectedgroups (FIG. 8A) but not for any of the group histopathological scoresfor the corresponding synovium in the right joints (FIG. 8B). The majorcontribution to the differences between control and MPC treated groupswould appear to be in the reduction in synovial hyperplasia in the celltreated groups (FIG. 8A).

The scores of synovial histopathological changes in the left and righthock joints of the intra-articular injected groups failed to show anysignificant differences between injection with saline or 25 million MPCbut as expected the scores for the left joints were higher than theright joint (FIG. 8C).

Example 7: Immunohistochemical Studies of the Synovial Tissues

The results of the scoring systems used to semi-quantify the cellularchanges and cytokine levels in the synovial tissues of the variousanimal groups following immunohistolochemical staining with theantibodies described in the methods section are shown in FIGS. 9A-9C.The use of 3 different methods of scoring these multiple sectionsprecluded the generation of a meaningful combined score for each of theexperimental groups examined. Therefore the mean scores obtained foreach of the experimental groups for the individual marker antibodiesused are presented separately and are shown in FIGS. 10A-10F.Representative photomicrograph examples of some these sections togetherwith the scores assigned are shown in FIGS. 11A-11E.

Although clear trends were apparent for a decrease in intimal IL-1 andVCAM-1 and an increase in Gamma-Delta TCR (FIGS. 10A-10C) nostatistically significant differences between the Control and MPCinjected groups could be demonstrated. However, synovial intima(synoviocyte) staining for IL-6 (p=0.029) and TNF-alpha (p=0.049) (FIG.10D) and interstitial tissues staining for CD-14 cells (FIG. 10E) forthe high MPC dose injected group was demonstrated to be significantlylower than the saline injected control group (p=0.009). Furthermore,levels of the cytokine IL-17 in the synovial interstitial tissues of thehigh dose group were also significantly lower than for the correspondingcontrol group (ANOVA, p<0.005) (FIG. 10F).

The results of the immunohistochemical scores obtained for the salineand MPC injected joints are shown in Table 14. There appeared to behigher staining for CD4, CD8 and CD79 in the MPC injected joints afinding which was consistent with the higher number of lymphocytesobserved in the MPC injected joints. However, the mean values forTNF-alpha and IL-17 in the MPC injected joints were lower than for thesaline injected joints, particularly for the intima region of the leftjoint synovium.

TABLE 14 Immunohistochemical scores for synovial tissues of hock jointsfrom RA animals injected intra-articularly with Saline or 25 millionMPC. CD4 CD8 γδ TCR CD79a CD14 Joint Tissue Left Left Right Right GroupSheep Left Right Left Right Left Right Left Right intima tissue intimatissue F(Saline) Y28 3 0 3 2 2 0 1 0 3 3 2 1 IA Y31 1 2 2 2 2 1 2 1 no 2no 1 intima intima Y30 1 1 1 2 1 0 1 1 3 1 2 1 Y34 2 1 1 1 1 1 1 0 no 3no 2 intima intima Y37 1 1 2 1 1 1 2 0 3 4 3 2 Y39 2 1 2 1 1 1 2 1 2 3 12 mean 1.67 1.00 1.83 1.50 1.33 0.67 1.50 0.50 2.75 2.67 2.00 1.50 SD0.82 0.63 0.75 0.55 0.52 0.52 0.55 0.55 0.50 1.03 0.82 0.55 E(MPC) Y26 40 4 0 2 0 2 0 2 2 2 1 IA Y27 1 0 2 1 1 1 1 0 2 3 3 1 Y29 4 1 4 1 2 2 2 12 3 2 1 Y32 2 1 1 1 0 1 3 0 2 2 2 2 Y38 2 1 3 2 2 1 2 2 2 2 4 2 Y40 4 24 1 2 1 3 0 3 3 2 1 mean 2.83 0.83 3.00 1.00 1.50 1.00 2.17 0.50 2.172.50 2.50 1.33 SD 1.33 0.75 1.26 0.63 0.84 0.63 0.75 0.84 0.41 0.55 0.840.52 VCAM-1 IL-6 Ki67 Factor VIII Joint Tissue Left Left Right RightLeft Left Right Right Left Left Right Right Group Sheep intima tissueintima tissue intima tissue intima tissue intima tissue intima tissueLeft Right F(Saline) Y28 0 1 1 0 no 0 0 0 no 3 0 0 23 27 IA intimaintima Y31 3 2 1 0 2 3 0 2 0 2 0 1 79 14 Y30 2 1 2 0 1 2 0 1 0 0 0 0 3420 Y34 no 1 no 0 no 4 no 1 no no 0 45 17 intima intima intima intimaintima intima Y37 2 1 1 1 0 1 0 0 0 1 0 0 59 36 Y39 1 1 1 0 1 3 0 1 0 0no 0 44 57 intima mean 1.60 1.17 1.20 0.17 1.00 2.17 0.00 0.83 0.00 1.200.00 0.17 47.03 28.39 SD 1.14 0.41 0.45 0.41 0.82 1.47 0.00 0.75 0.001.30 0.00 0.41 19.59 15.76 E(MPC) Y26 0 1 0 0 2 2 0 1 0 2 0 0 47 18 IAY27 2 1 2 0 1 1 0 1 0 1 0 0 80 22 Y29 2 1 2 0 1 3 0 3 2 0 39 32 Y32 1 01 1 0 3 0 2 0 0 0 0 29 28 Y38 2 0 1 0 0 2 0 1 0 1 0 0 36 32 Y40 3 2 2 11 2 1 1 0 45 40 mean 1.67 0.83 1.33 0.33 0.83 2.17 0.00 1.60 0.20 1.170.00 0.00 45.67 28.58 SD 1.03 0.75 0.82 0.52 0.75 0.75 0.00 0.89 0.450.75 0.00 0.00 18.02 7.85 TNF-a IL-17 IgM Joint Tissue Left Left RightRight Left Left Right Right Group Sheep intima tissue intima tissueintima tissue intima tissue F(Saline) Y28 no 0 1 0 no 3 1 0 IA intimaintima Y31 2 2 0 0 3 4 2 2 Y30 1 1 1 1 2 2 2 1 Y34 no 1 no 0 2 3 no 1intima intima intima Y37 0 0 0 0 3 3 1 1 Y39 1 1 1 0 2 3 1 1 mean 1.000.83 0.60 0.17 2.40 3.00 1.40 1.00 SD 0.82 0.75 0.55 0.41 0.55 0.63 0.550.63 E(MPC) Y26 0 0 0 0 1 1 0 1 IA Y27 0 0 0 0 1 2 2 0 Y29 1 1 0 0 2 3 11 Y32 0 1 0 0 1 2 1 1 Y38 1 0 0 0 2 3 2 3 Y40 1 0 3 3 2 2 mean 0.50 0.330.00 0.00 1.67 2.33 1.33 1.33 SD 0.55 0.52 0.00 0.00 0.82 0.82 0.82 1.03IL-10 IL-1b Joint Tissue Left Left Right Right Left Left Right RightGroup Sheep intima tissue intima tissue intima tissue intima tissueF(Saline) Y28 no 0 0 0 no 1 1 0 IA intima intima Y31 0 0 0 0 1 1 0 0 Y300 0 0 0 1 0 0 0 Y34 no 0 no 0 no 2 no 0 intima intima intima intima Y370 0 0 0 1 0 0 0 Y39 0 0 0 0 0 0 0 0 mean 0.00 0.00 0.00 0.00 0.75 0.670.20 0.00 SD 0.00 0.00 0.00 0.00 0.50 0.82 0.45 0.00 E(MPC) Y26 0 0 no 00 0 no 0 IA intima intima Y27 0 0 0 0 0 0 1 0 Y29 1 0 0 0 1 0 0 0 Y32 00 0 0 0 0 0 0 Y38 0 0 0 0 0 0 0 0 Y40 0 0 0 0 0 1 0 0 mean 0.17 0.000.00 0.00 0.17 0.17 0.20 0.00 SD 0.41 0.00 0.00 0.00 0.41 0.41 0.45 0.00L = Left joint, R = Right joint, LI = Left joint intima, RI = Rightintima. LT = Left interstitial Tissue, RT = Right interstitial Tissue

General Discussion

As disclosed herein, immunization of adult sheep by the SC injection ofbovine type II collagen in Freund's complete and incomplete adjuvant,followed by intra-articular (IA) injection of the same protein aloneinto their hock joints, resulted in the induction of an arthropathy thatdisplayed many of the classical pathological hallmarks of human RA.

This ovine model of RA was therefore considered to be suitable for theevaluation of the therapeutic effects of IV administered MPC at thedoses of 0.3×10⁶ cells/kg, 1.0×10⁶ cells/kg and 2.0×10⁶ cells/kgrelative to the effects of an equivalent volume of IV saline. Forcomparative purposes an IA group that received a single injection of25×10⁶ MPC or saline into the left hock joints was also included in thestudy. MPCs or saline were administered 14 days following theintra-articular injection of bovine type II collagen (day 28) andsacrificed 30 clays later (day 72).

The outcomes from the analysis of the histopathological andimmunohistochemical analysis of synovial tissues were positive.Moreover, of the three IV MPC doses administered, the highest dose of 2million MPC/kg consistently generated statistical improvements relativeto the saline control group. While the low dose MPC group also showedpositive effects in some of the parameters studied, the mid dose MPCtreated exhibited a mixed response. The reason for the absence of aclear dose response relationship in the present study is not entirelyclear but may be related to the small number of animals used in eachgroup and the heterogeneity in the intensity of disease expressionwithin the group as exemplified by the high standard deviations observedfor many of the control group parameters. Nevertheless, thestatistically significant reduction of histopathological scores observedfor the synovial membranes of the left hock joints from the high doseMPC groups relative to saline controls (FIG. 8C) were supported by thereduced immunohistochemical scores for synoviocyte (intima) staining forIL-6, TNF-alpha (FIG. 4D) and interstitial tissue staining for CD14cells (FIG. 10E) and IL-17 (FIG. 10F).

The results of the present studies using an ovine model of chronic RAhave confirmed that a single IV injection of between 0.3-2.0 millionMPC/kg was effective in reducing the key histopathological indices ofarthritis, namely, synovial hyperplasia, stromal tissue activation andinflammatory cell infiltration. Moreover the demonstration, usingimmunohistochemical staining of frozen sections, that the levels ofIL-6, TNF-alpha, IL-17 and CD14+ cells were significantly reduced in thehigh MPC dosed animals, relative to the saline controls was supportiveof our working hypothesis. IL-17 has been shown (Shahrara et al. (2009)Journal of Immunology 182: 3884-3891) to induce monocyte migration invivo leading to suggest that this cytokine was responsible for therecruitment of monocytes into the joints of patients with RA. This viewwas consistent with our hypothesis that by reducing the levels of IL-17in the synovial interstitium, the injected MPC indirectly diminished thenumbers of monocytes migrating from the bone marrow to the inflamedjoint thereby limiting the levels of pro-inflammatory cytokines, such asIL-6 and TNF-alpha produced by the synoviocytes of the proliferatingintima.

Moreover, “conventional” inflammatory cytokines expressed in cartilageand synovium have been suggested to play a role in osteoarthritis,including and interleukin-1β (IL-1 β), tumor necrosis factor alpha(TNFα), IL-6, IL-8, IL-17 and soluble CD14 (Liu-Bryan and TerkeltaubArthritis and Rheumatism, 64: 2055-2058, 2012). Thus, the data presentedherein support a role for the administration of MPCs (e.g.,intravenously) to treat osteoarthritis.

The invention claimed is:
 1. A method for treating a rheumatic diseasein a subject, the method comprising systemically administering to thesubject a therapeutically effective amount of a population of cellsenriched for STRO-1⁺ multipotential cells sufficient to reduce one ormore symptoms of rheumatic disease, thereby treating said rheumaticdisease in said subject.
 2. The method of claim 1, wherein the rheumaticdisease is selected from the group consisting of rheumatoid arthritis,Still's disease, ankylosing spondylitis, Reiter's disease, psoriatricarthritis, enteric arthritis, sacroiliitis, spondylitis andosteoarthritis.
 3. The method of claim 1, wherein the rheumatic diseaseis rheumatoid arthritis.
 4. The method of claim 1, wherein the subjectis receiving treatment with methotrexate prior to administration of thepopulation of cells enriched for STRO-1⁺ multipotential cells.
 5. Themethod of claim 1, wherein the STRO-1⁺ multipotential cells areSTRO-1^(bright) multipotential cells.
 6. The method of claim 1, whereinthe one or more symptoms are clinical features including one or more ofa reduced range of motion, joint swelling or tenderness, inflammationand pain.
 7. The method of claim 1, wherein said effective amount isbetween 0.3×10⁶ cells/kg to 2×10⁶ said cells/kg.
 8. The method of claim1, wherein the effective amount of the STRO-1⁺ multipotential cells isbetween about 1.5×10⁶ to about 4×10⁶ cells per kilogram.
 9. The methodof claim 1, wherein the effective amount of STRO-1⁺ multipotential cellsadministered to the subject is between about 100 million to about 300million cells.
 10. The method of claim 1, wherein the populationenriched for STRO-1⁺ multipotential cells are administered once weeklyor less often.
 11. The method of claim 1, wherein the populationenriched for STRO-1⁺ multipotential cells are administered once everyfour weeks or less often.
 12. The method of claim 1, wherein thepopulation enriched for STRO-1⁺ multipotential cells are autogenic orallogenic cells.
 13. The method of claim 1, wherein the populationenriched for STRO-1⁺ multipotential cells have been culture expandedprior to administration.
 14. The method of claim 1, wherein thepopulation enriched for STRO-1⁺ multipotential cells are STRO-1^(bright)and/or express tissue non-specific alkaline phosphatase (TNAP).
 15. Themethod according to claim 1, wherein the STRO-1⁺ multipotential cellsare administered in the form of a composition comprising said STRO-1⁺multipotential cells and a carrier and/or excipient.
 16. The method ofclaim 1, wherein evaluation criteria for reduced symptoms include arecognized disease activity score for a rheumatic disease.
 17. Themethod of claim 1, wherein administration is intravenous.